Nucleic acid and corresponding protein entitled 158P3D2 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 158P3D2) and its encoded protein, and variants thereof, are described wherein 158P3D2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 158P3D2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 158P3D2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 158P3D2 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Serial No. 60/283,112filed Apr. 10, 2001, and U.S. Serial No. 60/286,630, filed Apr. 25,2001. The contents of these applications are hereby incorporated byreference herein in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The invention described herein relates to a gene and its encodedprotein, termed 158P3D2 expressed in certain cancers, and to diagnosticand therapeutic methods and compositions useful in the management ofcancers that express 158P3D2.

BACKGROUND OF THE INVENTION

[0004] Cancer is the second leading cause of human death next tocoronary disease. Worldwide, millions of people die from cancer everyyear. In the United States alone, as reported by the American CancerSociety, cancer causes the death of well over a half-million peopleannually, with over 1.2 million new cases diagnosed per year. Whiledeaths from heart disease have been declining significantly, thoseresulting from cancer generally are on the rise. In the early part ofthe next century, cancer is predicted to become the leading cause ofdeath.

[0005] Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

[0006] Worldwide, prostate cancer is the fourth most prevalent cancer inmen. In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

[0007] On the diagnostic front, the lack of a prostate tumor marker thatcan accurately detect early-stage, localized tumors remains asignificant limitation in the diagnosis and management of this disease.Although the serum prostate specific antigen (PSA) assay has been a veryuseful tool, however its specificity and general utility is widelyregarded as lacking in several important respects.

[0008] Progress in identifying additional specific markers for prostatecancer has been improved by the generation of prostate cancer xenograftsthat can recapitulate different stages of the disease in mice. The LAPC(Los Angeles Prostate Cancer) xenografts are prostate cancer xenograftsthat have survived passage in severe combined immune deficient (SCID)mice and have exhibited the capacity to mimic the transition fromandrogen dependence to androgen independence (Klein et al., 1997, Nat.Med. 3:402). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin. CancerRes. September 1996 2 (9): 1445-51), STEAP (Hubert, et al., Proc NatlAcad Sci USA. Dec. 7, 1999; 96(25): 14523-8) and prostate stem cellantigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735).

[0009] While previously identified markers such as PSA, PSM, PCTA andPSCA have facilitated efforts to diagnose and treat prostate cancer,there is need for the identification of additional markers andtherapeutic targets for prostate and related cancers in order to furtherimprove diagnosis and therapy.

[0010] Renal cell carcinoma (RCC) accounts for approximately 3 percentof adult malignancies. Once adenomas reach a diameter of 2 to 3 cm,malignant potential exists. In the adult, the two principal malignantrenal tumors are renal cell adenocarcinoma and transitional cellcarcinoma of the renal pelvis or ureter. The incidence of renal celladenocarcinoma is estimated at more than 29,000 cases in the UnitedStates, and more than 11,600 patients died of this disease in 1998.Transitional cell carcinoma is less frequent, with an incidence ofapproximately 500 cases per year in the United States.

[0011] Surgery has been the primary therapy for renal celladenocarcinoma for many decades. Until recently, metastatic disease hasbeen refractory to any systemic therapy. With recent developments insystemic therapies, particularly immunotherapies, metastatic renal cellcarcinoma may be approached aggressively in appropriate patients with apossibility of durable responses. Nevertheless, there is a remainingneed for effective therapies for these patients.

[0012] Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

[0013] Most bladder cancers recur in the bladder. Bladder cancer ismanaged with a combination of transurethral resection of the bladder(TUR) and intravesical chemotherapy or immunotherapy. The multifocal andrecurrent nature of bladder cancer points out the limitations of TUR.Most muscle-invasive cancers are not cured by TUR alone. Radicalcystectomy and urinary diversion is the most effective means toeliminate the cancer but carry an undeniable impact on urinary andsexual function. There continues to be a significant need for treatmentmodalities that are beneficial for bladder cancer patients.

[0014] An estimated 130,200 cases of colorectal cancer occurred in 2000in the United States, including 93,800 cases of colon cancer and 36,400of rectal cancer. Colorectal cancers are the third most common cancersin men and women. Incidence rates declined significantly during1992-1996 (-2.1% per year). Research suggests that these declines havebeen due to increased screening and polyp removal, preventingprogression of polyps to invasive cancers. There were an estimated56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in2000, accounting for about 11% of all U.S. cancer deaths.

[0015] At present, surgery is the most common form of therapy forcolorectal cancer, and for cancers that have not spread, it isfrequently curative. Chemotherapy, or chemotherapy plus radiation, isgiven before or after surgery to most patients whose cancer has deeplyperforated the bowel wall or has spread to the lymph nodes. A permanentcolostomy (creation of an abdominal opening for elimination of bodywastes) is occasionally needed for colon cancer and is infrequentlyrequired for rectal cancer. There continues to be a need for effectivediagnostic and treatment modalities for colorectal cancer.

[0016] There were an estimated 164,100 new cases of lung and bronchialcancer in 2000, accounting for 14% of all U.S. cancer diagnoses. Theincidence rate of lung and bronchial cancer is declining significantlyin men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the1990s, the rate of increase among women began to slow. In 1996, theincidence rate in women was 42.3 per 100,000.

[0017] Lung and bronchial cancer caused an estimated 156,900 deaths in2000, accounting for 28% of all cancer deaths. During 1992-1996,mortality from lung cancer declined significantly among men (−1.7% peryear) while rates for women were still significantly increasing (0.9%per year). Since 1987, more women have died each year of lung cancerthan breast cancer, which, for over 40 years, was the major cause ofcancer death in women. Decreasing lung cancer incidence and mortalityrates most likely resulted from decreased smoking rates over theprevious 30 years; however, decreasing smoking patterns among women lagbehind those of men. Of concern, although the declines in adult tobaccouse have slowed, tobacco use in youth is increasing again.

[0018] Treatment options for lung and bronchial cancer are determined bythe type and stage of the cancer and include surgery, radiation therapy,and chemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

[0019] An estimated 182,800 new invasive cases of breast cancer wereexpected to occur among women in the United States during 2000.Additionally, about 1,400 new cases of breast cancer were expected to bediagnosed in men in 2000. After increasing about 4% per year in the1980s, breast cancer incidence rates in women have leveled off in the1990s to about 110.6 cases per 100,000.

[0020] In the U.S. alone, there were an estimated 41,200 deaths (40,800women, 400 men) in 2000 due to breast cancer. Breast cancer ranks secondamong cancer deaths in women. According to the most recent data,mortality rates declined significantly during 1992-1996 with the largestdecreases in younger women, both white and black. These decreases wereprobably the result of earlier detection and improved treatment.

[0021] Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

[0022] Local excision of ductal carcinoma in situ (DCIS) with adequateamounts of surrounding normal breast tissue may prevent the localrecurrence of the DCIS. Radiation to the breast and/or tamoxifen mayreduce the chance of DCIS occurring in the remaining breast tissue. Thisis important because DCIS, if left untreated, may develop into invasivebreast cancer. Nevertheless, there are serious side effects or sequelaeto these treatments. There is, therefore, a need for efficacious breastcancer treatments.

[0023] There were an estimated 23,100 new cases of ovarian cancer in theUnited States in 2000. It accounts for 4% of all cancers among women andranks second among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

[0024] Surgery, radiation therapy, and chemotherapy are treatmentoptions for ovarian cancer. Surgery usually includes the removal of oneor both ovaries, the fallopian tubes (salpingo-oophorectomy), and theuterus (hysterectomy). In some very early tumors, only the involvedovary will be removed, especially in young women who wish to havechildren. In advanced disease, an attempt is made to remove allintra-abdominal disease to enhance the effect of chemotherapy. Therecontinues to be an important need for effective treatment options forovarian cancer.

[0025] There were an estimated 28,300 new cases of pancreatic cancer inthe United States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

[0026] Surgery, radiation therapy, and chemotherapy are treatmentoptions for pancreatic cancer. These treatment options can extendsurvival and/or relieve symptoms in many patients but are not likely toproduce a cure for most. There is a significant need for additionaltherapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

[0027] The present invention relates to a gene, designated 158P3D2, thathas now been found to be overexpressed in the cancer(s) listed in TableI. Northern blot expression analysis of 158P3D2 gene expression innormal tissues shows a restricted expression pattern in adult tissues.The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of158P3D2 are provided. The tissue-related profile of 158P3D2 in normaladult tissues, combined with the over-expression observed in the tumorslisted in Table I, shows that 158P3D2 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

[0028] The invention provides polynucleotides corresponding orcomplementary to all or part of the 158P3D2 genes, mRNAs, and/or codingsequences, preferably in isolated form, including polynucleotidesencoding 158P3D2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300,325, 328 or more than 328 contiguous amino acids of a 158P3D2-relatedprotein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNAhybrids, and related molecules, polynucleotides or oligonucleotidescomplementary or having at least a 90% homology to the 158P3D2 genes ormRNA sequences or parts thereof, and polynucleotides or oligonucleotidesthat hybridize to the 158P3D2 genes, mRNAs, or to 158P3D2-encodingpolynucleotides. Also provided are means for isolating cDNAs and thegenes encoding 158P3D2. Recombinant DNA molecules containing 158P3D2polynucleotides, cells transformed or transduced with such molecules,and host-vector systems for the expression of 158P3D2 gene products arealso provided. The invention further provides antibodies that bind to158P3D2 proteins and polypeptide fragments thereof, including polyclonaland monoclonal antibodies, murine and other mammalian antibodies,chimeric antibodies, humanized and fully human antibodies, andantibodies labeled with a detectable marker or therapeutic agent. Incertain embodiments there is a proviso that the entire nucleic acidsequence of FIG. 2 is not encoded and/or the entire amino acid sequenceof FIG. 2 is not prepared. In certain embodiments, the entire nucleicacid sequence of FIG. 2 is encoded and/or the entire amino acid sequenceof FIG. 2 is prepared, either of which are in respective human unit doseforms.

[0029] The invention further provides methods for detecting the presenceand status of 158P3D2 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 158P3D2.A typical embodiment of this invention provides methods for monitoring158P3D2 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

[0030] The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 158P3D2such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of158P3D2 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 158P3D2 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of158P3D2. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 158P3D2 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

[0031] In another aspect, the agent comprises one or more than onepeptide which comprises a cytotoxic T lymphocyte (CTL) epitope thatbinds an HLA class I molecule in a human to elicit a CTL response to158P3D2 and/or one or more than one peptide which comprises a helper Tlymphocyte (HTL) epitope which binds an HLA class II molecule in a humanto elicit an HTL response. The peptides of the invention may be on thesame or on one or more separate polypeptide molecules. In a furtheraspect of the invention, the agent comprises one or more than onenucleic acid molecule that expresses one or more than one of the CTL orHTL response stimulating peptides as described above. In yet anotheraspect of the invention, the one or more than one nucleic acid moleculemay express a moiety that is immunologically reactive with 158P3D2 asdescribed above. The one or more than one nucleic acid molecule may alsobe, or encodes, a molecule that inhibits production of 158P3D2.Non-limiting examples of such molecules include, but are not limited to,those complementary to a nucleotide sequence essential for production of158P3D2 (e.g. antisense sequences or molecules that form a triple helixwith a nucleotide double helix essential for 158P3D2 production) or aribozyme effective to lyse 158P3D2 mRNA.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1. The 158P3D2 SSH sequence of 312 nucleotides.

[0033]FIG. 2. The cDNA (SEQ ID. NO.: ______) and amino acid sequence(SEQ ID. NO.: ______) of 158P3D2 variant 1 clone 158P3D2-BCP1 (alsocalled “158P3D2 v.1” or “158P3D2 variant 1” or “158P3D2 var1”) is shownin FIG. 2A. The start methionine is underlined. The open reading frameextends from nucleic acid 849-1835 including the stop codon. The cDNA(SEQ ID. NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of158P3D2 variant 2a (also called “158P3D2 var2a” or “158P3D2 v.2a”) isshown in FIG. 2B. The codon for the start methionine is underlined. Theopen reading frame extends from nucleic acid 117 to 827 including thestop codon. The cDNA (SEQ ID. NO. : ______) and amino acid sequence (SEQID. NO.: ______) of 158P3D2 variant 2b (also called “158P3D2 var2b” or“158P3D2 v.2b”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid2249-2794 including the stop codon. The cDNA (SEQ ID. NO.: ______) andamino acid sequence (SEQ ID. NO.: ______) of 158P3D2 variant 3 (alsocalled “158P3D2 var3” or “158P3D2 v.3”) is shown in FIG. 2D. The codonfor the start methionine is underlined. The open reading frame extendsfrom nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID.NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of 158P3D2variant 4 (also called “158P3D2 var4” or “158P3D2 v.4”) is shown in FIG.2E. The codon for the start methionine is underlined. The open readingframe extends from nucleic acid 849-1835 including the stop codon. ThecDNA (SEQ ID. NO.: ______) and amino acid sequence (SEQ ID. NO.: ______)of 158P3D2 variant 5a clone 158P3D2-BCP2 (also called “158P3D2 variantSa” or “158P3D2 varSa” or “158P3D2 v.5a”) is shown in FIG. 2F. The codonfor the start methionine is underlined. The open reading frame extendsfrom nucleic acid 849-1385 including the stop codon. The cDNA (SEQ ID.NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of 158P3D2variant 5b clone 158P3D2-BCP2 (also called “158P3D2 variant Sb” or“158P3D2 var5b” or “158P3D2 v.Sb”) is shown in FIG. 2G. The codon forthe start methionine is underlined. The open reading frame extends fromnucleic acid 1289-1834 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.: ______) of 158P3D2 variant6 (also called “158P3D2 var6” or “158P3D2 v.6”) is shown in FIG. 2H. Thecodon for the start methionine is underlined. The open reading frameextends from nucleic acid 849-1835 including the stop codon. The cDNA(SEQ ID. NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of158P3D2 variant 7 (also called “158P3D2 var7” or “158P3D2 v.7”) is shownin FIG. 2I. The codon for the start methionine is underlined. The openreading frame extends from nucleic acid 849-1835 including the stopcodon. The cDNA (SEQ ID. NO.: ______) and amino acid sequence (SEQ ID.NO.: ______) of 158P3D2 variant 8 (also called “158P3D2 var8” or“158P3D2 v.8”) is shown in FIG. 2J. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 849-1835including the stop codon. As used herein, a reference to 158P3D2includes all variants thereof, including those shown in FIG. 10.

[0034]FIG. 3. Amino acid sequence of 158P3D2 var1 (SEQ ID. NO.: ______)is shown in FIG. 3A; it has 328 amino acids. The amino acid sequence of158P3D2 var2a (SEQ ID. NO.: ______) is shown in FIG. 3B; it has 236amino acids. The amino acid sequence of 158P3D2 var2b (SEQ ID. NO.:______) is shown in FIG. 3C; it has 181 amino acids. The amino acidsequence of 158P3D2 var3 (SEQ ID. NO.: ______) is shown in FIG. 3D; ithas 328 amino acids. The amino acid sequence of 158P3D2 var4 (SEQ ID.NO.: ______) is shown in FIG. 3E; it has 328 amino acids. The amino acidsequence of 158P3D2 var5a (SEQ ID. NO.: ______) is shown in FIG. 3F; ithas 178 amino acids. The amino acid sequence of 158P3D2 var5b (SEQ ID.NO.: ______) is shown in FIG. 3G; it has 181 amino acids. As usedherein, a reference to 158P3D2 includes all variants thereof, includingthose shown in FIG. 11.

[0035]FIG. 4. The nucleic acid sequence alignment of 158P3D2 var1 tofer-1-like 4 (C.elegans) (FER1L4) mRNA is shown in FIG. 4A. The aminoacid sequence alignment of 158P3D2 var1 to dJ47704.1.1 (AL121586), anovel protein similar to otoferlin and dysferlin, isoform 1 is shown inFIG. 4B. The amino acid sequence alignment with human brain otoferlinlong isoform is shown in FIG. 4C. The amino acid sequence alignment withmouse otoferlin is shown in FIG. 4D. The amino acid sequence alignmentsof 158P3D2 protein var1, 2a, 2b, 3, 4, 5a, and 5b are shown in FIG. 4E.

[0036]FIG. 5. Hydrophilicity amino acid profile of A) 158P3D2 var1, B)158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithmsequence analysis using the method of Hopp and Woods (Hopp T. P., WoodsK. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on theProtscale website (www.expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

[0037]FIG. 6. Hydropathicity amino acid profile of A) 158P3D2 var1, B)158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithmsequence analysis using the method of Kyte and Doolittle (Kyte J.,Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on theProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

[0038]FIG. 7. Percent accessible residues amino acid profile of A)158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined bycomputer algorithm sequence analysis using the method of Janin (JaninJ., 1979 Nature 277:491-492) accessed on the ProtScale website(www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

[0039]FIG. 8. Average flexibility amino acid profile of A) 158P3D2 var1,B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithmsequence analysis using the method of Bhaskaran and Ponnuswamy(Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.32:242-255) accessed on the ProtScale website(www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

[0040]FIG. 9. Beta-turn amino acid profile of A) 158P3D2 var1, B)158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithmsequence analysis using the method of Deleage and Roux (Deleage, G.,Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScalewebsite (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasymolecular biology server.

[0041]FIG. 10. Schematic display of nucleotide variants of 158P3D2.Variant 158P3D2 v.2 is an alternative transcript. Others are SingleNucleotide Polymorphism (also called “SNP”) variants, which could alsooccur in any alternative transcript. The numbers in “( )” underneath thebox correspond to those of 158P3D2 var1. ‘-’ indicate single nucleotidedeletion. Variants 158P3D2 v.3 through v.8 are variants with singlenucleotide variations. The black boxes show the same sequence as 158P3D2var1. SNPs are indicated above the box.

[0042]FIG. 11. Schematic display of protein variants of 158P3D2.Nucleotide variant 158P3D2 var2 and 158P3D2 v.5 in FIG. 10 potentiallycode for two different proteins, designated as variants 158P3D2 var2aand 158P3D2 var2b, 158P3D2 v.5a and 158P3D2 v.5b, respectively. Variant158P3D2 v.5b shares the same amino acid sequence as variant 158P3D2var2b. Variants 158P3D2 v.3 and v.4 are variants with single amino acidvariations. The black boxes show the same sequence as 158P3D2 var1. Thenumbers in “( )” underneath the box correspond to those of 158P3D2 var1.Single amino acid differences are indicated above the box.

[0043]FIG. 12. Secondary structure prediction of 158P3D2 var1 (FIG.12A), var2a (FIG. 12B) and var5a (FIG. 12C); and transmembranepredictions for 158P3D2 var1 (FIGS. 12D and E). The secondary structureof 158P3D2 proteins were predicted using the HNN—Hierarchial NeuralNetwork method (Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).This method predicts the presence and location of alpha helices,extended strands, and random coils from the primary protein sequence.The percent of the protein in a given secondary structure is also given.

[0044] A schematic representation of the probability of existence oftransmembrane regions and orientation based on the TMpred algorithmwhich utilizes TMBASE is shown in FIG. 12D (K. Hofmann, W. Stoffel.TMBASE—A database of membrane spanning protein segments Biol. Chem.Hoppe-Seyler 374:166, 1993). A schematic representation of theprobability of the existence of transmembrane regions and theextracellular and intracellular orientation based on the TMHMM algorithmis shown in FIG. 12E (Erik L. L. Sonnhammer, Gunnar von Heijne, andAnders Krogh: A hidden Markov model for predicting transmembrane helicesin protein sequences. In Proc. of Sixth Int. Conf. on IntelligentSystems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn,F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAIPress, 1998). The TMpred and TMHMM algorithms are accessed from theExPasy molecular biology server (http://www.expasy.ch/tools/). Theresults of the transmembrane prediction programs depict 158P3D2 var1 ascontaining 1 transmembrane domain.

[0045]FIG. 13. Exon compositions of transcript variants of 158P3D2.Variant 158P3D2 var2 is an alternative transcript. Compared with 158P3D2var1, it has six additional exons to the 5′ end, an exon 7 longer thanexon 1 of 158P3D2 var1 and an exon 10 shorter than exon 4 of 158P3D2var. Exons 2, 3, 5, 6 and 7 of 158P3D2 var1 are the same as exons 8, 9,11, 12 and 13 of 158P3D2 var2, respectively. The numbers in “( )”underneath the box correspond to those of 158P3D2 var1. The black boxesshow the same sequence as 158P3D2 var1. The length of the introns arenot proportional.

[0046]FIG. 14. Expression of 158P3D2 by RT-PCR. First strand cDNA wasprepared from vital pool 1 (liver, lung and kidney), vital pool 2(pancreas, colon and stomach), prostate cancer metastasis to lymph nodefrom 2 different patients, prostate cancer pool, bladder cancer pool,kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancerpool, breast cancer pool, cancer metastasis pool, and pancreas cancerpool. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at26 and 30 cycles of amplification. Results show strong expression of158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasispool. Expression of 158P3D2 is also detected in colon cancer pool, lungcancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pooland prostate metastases to lymph node, and vital pool 2, but not vitalpool 1.

[0047]FIG. 15. Expression of 158P3D2 in normal tissues. Two multipletissue northern blots (Clontech) both with 2 ug of mRNA/lane were probedwith the 158P3D2 SSH fragment. Size standards in kilobases (kb) areindicated on the side. Results show restricted expression of anapproximately 8 kb 158P3D2 transcript in normal placenta.

[0048]FIG. 16. Expression of 158P3D2 in Multiple Normal Tissues. An mRNAdot blot containing 76 different samples from human tissues was analyzedusing a 158P3D2 probe. Expression was detected in placenta and stomach.

[0049]FIG. 17. Expression of 158P3D2 in Patient Cancer Specimens andNormal Tissues. RNA was extracted from a pool of three bladder cancers,as well as from normal prostate (NP), normal bladder (NB), normal kidney(NK), normal colon (NC), normal lung (NL) and normal breast (NBr).Northern blot with 10 10 μg of total RNA/lane was probed with 158P3D2sequence. Size standards in kilobases (kb) are indicated on the side.The results show expression of 158P3D2 in the bladder cancer pool butnot in the normal tissues tested.

[0050]FIG. 18. Expression of 158P3D2 in bladder cancer patient tissues.RNA was extracted from normal bladder (N), bladder cancer cell lines(UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and theirnormal adjacent tissues (NAT). Northern blots with 10 ug of total RNAwere probed with the 158P3D2 SSH fragment. Size standards in kilobasesare on the side. Results show strong expression of 158P3D2 in tumortissues. The expression observed in normal adjacent tissue (isolatedfrom diseased tissues) but not in normal tissue, isolated from healthydonors, may indicate that these tissues are not fully normal and that158P3D2 may be expressed in early stage tumors.

[0051]FIG. 19. 158P3D2 Expression in 293T Cells Following Transfection.293T cells were transfected with either 158P3D2.pcDNA3.1/mychis orpcDNA3.1/mychis vector control. Forty hours later, cell lysates werecollected. Samples were run on an SDS-PAGE acrylamide gel, blotted andstained with anti-his antibody. The blot was developed using the ECLchemiluminescence kit and visualized by autoradiography. Results showexpression of 158P3D2 clones of 158P3D2.pcDNA3.1/mychis in the lysatesof 158P3D2 .pcDNA3. 1/mychis transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Outline of Sections

[0053] I.) Definitions

[0054] II.) 158P3D2 Polynucleotides

[0055] II.A.) Uses of 158P3D2 Polynucleotides

[0056] II.A.1.) Monitoring of Genetic Abnormalities

[0057] II.A.2.) Antisense Embodiments

[0058] II.A.3.) Primers and Primer Pairs

[0059] II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid Molecules

[0060] II.A.5.) Recombinant Nucleic Acid Molecules and Host-VectorSystems

[0061] III.) 158P3D2-Related Proteins

[0062] III.A.) Motif-bearing Protein Embodiments

[0063] III.B.) Expression of 158P3D2-Related Proteins

[0064] III.C.) Modifications of 158P3D2-Related Proteins

[0065] III.D.) Uses of 158P3D2-Related Proteins

[0066] IV.) 158P3D2 Antibodies

[0067] V.) 158P3D2 Cellular Immune Responses

[0068] VI.) 158P3D2 Transgenic Animals

[0069] VII.) Methods for the Detection of 158P3D2

[0070] VIII.) Methods for Monitoring the Status of 158P3D2-Related Genesand Their Products

[0071] IX.) Identification of Molecules that Interact with 158P3D2

[0072] X.) Therapeutic Methods and Compositions

[0073] X.A.) Anti-Cancer Vaccines

[0074] X.B.) 158P3D2 as a Target for Antibody-Based Therapy

[0075] X.C.) 158P3D2 as a Target for Cellular Immune Responses

[0076] X.C.1. Minigene Vaccines

[0077] X.C.2. Combinations of CTL Peptides with Helper Peptides

[0078] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

[0079] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/orHTL Peptides

[0080] X.D.) Adoptive Immunotherapy

[0081] X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

[0082] XI.) Diagnostic and Prognostic Embodiments of 158P3D2.

[0083] XII.) Inhibition of 158P3D2 Protein Function

[0084] XII.A.) Inhibition of 158P3D2 with Intracellular Antibodies

[0085] XII.B.) Inhibition of 158P3D2 with Recombinant Proteins

[0086] XII.C.) Inhibition of 158P3D2 Transcription or Translation

[0087] XII.D.) General Considerations for Therapeutic Strategies

[0088] XIII.) KITS

[0089] I.) Definitions:

[0090] Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

[0091] The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

[0092] “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence 158P3D2 (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 158P3D2. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

[0093] The term “analog” refers to a molecule which is structurallysimilar or shares similar or corresponding attributes with anothermolecule (e.g. a 158P3D2-related protein). For example an analog of a158P3D2 protein can be specifically bound by an antibody or T cell thatspecifically binds to 158P3D2.

[0094] The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-158P3D2antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

[0095] An “antibody fragment” is defined as at least a portion of thevariable region of the immunoglobulin molecule that binds to its target,i.e., the antigen-binding region. In one embodiment it specificallycovers single anti-158P3D2 antibodies and clones thereof (includingagonist, antagonist and neutralizing antibodies) and anti-1 58P3D2antibody compositions with polyepitopic specificity.

[0096] The term “codon optimized sequences” refers to nucleotidesequences that have been optimized for a particular host species byreplacing any codons having a usage frequency of less than about 20%.Nucleotide sequences that have been optimized for expression in a givenhost species by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

[0097] The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

[0098] The term “homolog” refers to a molecule which exhibits homologyto another molecule, by for example, having sequences of chemicalresidues that are the same or similar at corresponding positions.

[0099] “Human Leukocyte Antigen” or “HLA” is a human class I or class IIMajor Histocompatibility Complex (MHC) protein (see, e.g., Stites, etal., Immunology, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

[0100] The terms “hybridize”, “hybridizing”, “hybridizes” and the like,used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C.

[0101] The phrases “isolated” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany the material as it is found in its native state.Thus, isolated peptides in accordance with the invention preferably donot contain materials normally associated with the peptides in their insitu environment. For example, a polynucleotide is said to be “isolated”when it is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 158P3D2 genes orthat encode polypeptides other than 158P3D2 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 158P3D2 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 158P3D2 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 158P3D2 protein. Alternatively, an isolated proteincan be prepared by chemical means.

[0102] The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

[0103] The terms “metastatic prostate cancer” and “metastatic disease”mean prostate cancers that have spread to regional lymph nodes or todistant sites, and are meant to include stage D disease under the AUAsystem and stage TxNxM+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is a preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation. Approximately half of theseandrogen-refractory patients die within 6 months after developing thatstatus. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are often osteoblastic rather thanosteolytic (i.e., resulting in net bone formation). Bone metastases arefound most frequently in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites for metastasis include lymphnodes, lung, liver and brain. Metastatic prostate cancer is typicallydiagnosed by open or laparoscopic pelvic lymphadenectomy, whole bodyradionuclide scans, skeletal radiography, and/or bone lesion biopsy.

[0104] The term “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts.

[0105] A “motif”, as in biological motif of an 158P3D2-related protein,refers to any pattern of amino acids forming part of the primarysequence of a protein, that is associated with a particular function(e.g. protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

[0106] A “pharmaceutical excipient” comprises a material such as anadjuvant, a carrier, pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservative, and the like.

[0107] “Pharmaceutically acceptable” refers to a non-toxic, inert,and/or composition that is physiologically compatible with humans orother mammals.

[0108] The term “polynucleotide” means a polymeric form of nucleotidesof at least 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

[0109] The term “polypeptide” means a polymer of at least about 4, 5, 6,7, or 8 amino acids. Throughout the specification, standard three letteror single letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

[0110] An HLA “primary anchor residue” is an amino acid at a specificposition along a peptide sequence which is understood to provide acontact point between the immunogenic peptide and the HLA molecule. Oneto three, usually two, primary anchor residues within a peptide ofdefined length generally defines a “motif” for an immunogenic peptide.These residues are understood to fit in close contact with peptidebinding groove of an HLA molecule, with their side chains buried inspecific pockets of the binding groove. In one embodiment, for example,the primary anchor residues for an HLA class I molecule are located atposition 2 (from the amino terminal position) and at the carboxylterminal position of a 8, 9, 10, 11, or 12 residue peptide epitope inaccordance with the invention. In another embodiment, for example, theprimary anchor residues of a peptide that will bind an HLA class IImolecule are spaced relative to each other, rather than to the terminiof a peptide, where the peptide is generally of at least 9 amino acidsin length. The primary anchor positions for each motif and supermotifare set forth in Table IV. For example, analog peptides can be createdby altering the presence or absence of particular residues in theprimary and/or secondary anchor positions shown in Table IV. Suchanalogs are used to modulate the binding affinity and/or populationcoverage of a peptide comprising a particular HLA motif or supermotif.

[0111] A “recombinant” DNA or RNA molecule is a DNA or RNA molecule thathas been subjected to molecular manipulation in vitro.

[0112] Non-limiting examples of small molecules include compounds thatbind or interact with 158P3D2, ligands including hormones,neuropeptides, chemokines, odorants, phospholipids, and functionalequivalents thereof that bind and preferably inhibit 158P3D2 proteinfunction. Such non-limiting small molecules preferably have a molecularweight of less than about 10 kDa, more preferably below about 9, about8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments,small molecules physically associate with, or bind, 158P3D2 protein; arenot found in naturally occurring metabolic pathways; and/or are moresoluble in aqueous than non-aqueous solutions

[0113] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0114] “Stringent conditions” or “high stringency conditions”, asdefined herein, are identified by, but not limited to, those that: (1)employ low ionic strength and high temperature for washing, for example0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent,such as formamide, for example, 50% (v/v) formamide with 0.1% bovineserum albumin/0. 1% Ficoll/0. 1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 1 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C. “Moderately stringent conditions” are described by, butnot limited to, those in Sambrook et al., Molecular Cloning: ALaboratory Manual, New York: Cold Spring Harbor Press, 1989, and includethe use of washing solution and hybridization conditions (e.g.,temperature, ionic strength and % SDS) less stringent than thosedescribed above. An example of moderately stringent conditions isovernight incubation at 37° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mLdenatured sheared salmon sperm DNA, followed by washing the filters in1×SSC at about 37-50° C. The skilled artisan will recognize how toadjust the temperature, ionic strength, etc. as necessary to accommodatefactors such as probe length and the like.

[0115] An HLA “supermotif” is a peptide binding specificity shared byHLA molecules encoded by two or more HLA alleles.

[0116] As used herein “to treat” or “therapeutic” and grammaticallyrelated terms, refer to any improvement of any consequence of disease,such as prolonged survival, less morbidity, and/or a lessening of sideeffects which are the byproducts of an alternative therapeutic modality;full eradication of disease is not required.

[0117] A “transgenic animal” (e.g., a mouse or rat) is an animal havingcells that contain a transgene, which transgene was introduced into theanimal or an ancestor of the animal at a prenatal, e.g., an embryonicstage. A “transgene” is a DNA that is integrated into the genome of acell from which a transgenic animal develops.

[0118] As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-328 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, or 328 or morepeptides of the invention. The peptides or polypeptides can optionallybe modified, such as by lipidation, addition of targeting or othersequences. HLA class I peptides of the invention can be admixed with, orlinked to, HLA class II peptides, to facilitate activation of bothcytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can alsocomprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

[0119] The term “variant” refers to a molecule that exhibits a variationfrom a described type or norm, such as a protein that has one or moredifferent amino acid residues in the corresponding position(s) of aspecifically described protein (e.g. the 158P3D2 protein shown in FIG. 2or FIG. 3. An analog is an example of a variant protein. Splice isoformsand SNPs are further examples of variants.

[0120] The “158P3D2-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 158P3D2 proteins orfragments thereof, as well as fusion proteins of a 158P3D2 protein and aheterologous polypeptide are also included. Such 158P3D2 proteins arecollectively referred to as the 158P3D2-related proteins, the proteinsof the invention, or 158P3D2. The term “158P3D2-related protein” refersto a polypeptide fragment or an 158P3D2 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 328 or more than 328 amino acids.

[0121] II.) 158P3D2 Polynucleotides

[0122] One aspect of the invention provides polynucleotidescorresponding or complementary to all or part of an 158P3D2 gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding an 158P3D2-related protein and fragmentsthereof, DNA, RNA, DNA/RNA hybrid, and related molecules,polynucleotides or oligonucleotides complementary to an 158P3D2 gene ormRNA sequence or a part thereof, and polynucleotides or oligonucleotidesthat hybridize to an 158P3D2 gene, mRNA, or to an 158P3D2 encodingpolynucleotide (collectively, “158P3D2 polynucleotides”). In allinstances when referred to in this section, T can also be U in FIG. 2.

[0123] Embodiments of a 158P3D2 polynucleotide include: a 158P3D2polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 158P3D2 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 158P3D2 nucleotides comprise, without limitation:

[0124] (I) a polynucleotide comprising, consisting essentially of, orconsisting of a sequence as shown in FIG. 2A (SEQ ID NO: ______),wherein T can also be U;

[0125] (II) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2A (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0126] (III) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2B (SEQ ID NO: ______), fromnucleotide residue number 117 through nucleotide residue number 827,including the stop codon, wherein T can also be U;

[0127] (IV) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2C (SEQ ID NO: ______), fromnucleotide residue number 2249 through nucleotide residue number 2794,including the a stop codon, wherein T can also be U;

[0128] (V) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2D (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0129] (VI) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2E (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0130] (VII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2F (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1385,including the stop codon, wherein T can also be U;

[0131] (VIII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2G (SEQ ID NO: ______), fromnucleotide residue number 1289 through nucleotide residue number 1834,including the stop codon, wherein T can also be U;

[0132] (IX) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2H (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0133] (X) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2I (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0134] (XI) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2J (SEQ ID NO: ______), fromnucleotide residue number 849 through nucleotide residue number 1835,including the stop codon, wherein T can also be U;

[0135] (XII) a polynucleotide that encodes an 158P3D2-related proteinthat is at least 90% homologous to an entire amino acid sequence shownin FIGS. 2A-I (SEQ ID NO: ______);

[0136] (XIII) a polynucleotide that encodes an 158P3D2-related proteinthat is at least 90% identical to an entire amino acid sequence shown inFIGS. 2A-I (SEQ ID NO: ______);

[0137] (XIV) a polynucleotide that encodes at least one peptide setforth in Tables V-XIX;

[0138] (XV) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIG. 3A in any whole number increment up to328 that includes an amino acid position having a value greater than 0.5in the Hydrophilicity profile of FIG. 5A; or of FIG. 3B in any wholenumber increment up to 236 that includes an amino acid position having avalue greater than 0.5 in the Hydrophilicity profile of FIG. 5B; or FIG.3F in any whole number increment up to 178 that includes an amino acidposition having a value greater than 0.5 in the Hydrophilicity profileof FIG. 5C;

[0139] (XVI) a polynucleotide that encodes a peptide region of at least5 amino acids of a peptide of FIG. 3A in any whole number increment upto 328 that includes an amino acid position having a value less than 0.5in the Hydropathicity profile of FIG. 6A; or of FIG. 3B in any wholenumber increment up to 236, that includes an amino acid position havinga value less than 0.5 in the Hydropathicity profile of FIG. 6B; or FIG.3F in any whole number increment up to 178 that includes an amino acidposition having a value greater than 0.5 in the Hydropathicity profileof FIG. 6C;

[0140] (XVII) a polynucleotide that encodes a peptide region of at least5 amino acids of a peptide of FIG. 3A in any whole number increment upto 328 that includes an amino acid position having a value greater than0.5 in the Percent Accessible Residues profile of FIG. 7A; or of FIG. 3Bin any whole number increment up to 236, that includes an amino acidposition having a value greater than 0.5 in the Percent AccessibleResidues profile of FIG. 7B; or FIG. 3F in any whole number increment upto 178 that includes an amino acid position having a value greater than0.5 in the Percent Accessible Residues profile of FIG. 7C;

[0141] (XVIII) a polynucleotide that encodes a peptide region of atleast 5 amino acids of a peptide of FIG. 3A in any whole numberincrement up to 328 that includes an amino acid position having a valuegreater than 0.5 in the Average Flexibility profile on FIG. 8A; or ofFIG. 3B in any whole number increment up to 236, that includes an aminoacid position having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8B; or FIG. 3F in any whole number increment up to 178that includes an amino acid position having a value greater than 0.5 inthe Average Flexibility profile of FIG. 8C;

[0142] (XIX) a polynucleotide that encodes a peptide region of at least5 amino acids of a peptide of FIG. 3A in any whole number increment upto 328 that includes an amino acid position having a value greater than0.5 in the Beta-turn profile of FIG. 9A; or of FIG. 3B in any wholenumber increment up to 236, that includes an amino acid position havinga value greater than 0.5 in the Beta-turn profile of FIG. 9B; or FIG. 3Fin any whole number increment up to 178 that includes an amino acidposition having a value greater than 0.5 in the Beta-turn profile ofFIG. 9C;

[0143] (XX) a polynucleotide that is fully complementary to apolynucleotide of any one of (I)-(XIX).

[0144] (XXI) a peptide that is encoded by any of (I)-(XX); and

[0145] (XXII) a polynucleotide of any of (I)-(XX) or peptide of (XXI)together with a pharmaceutical excipient and/or in a human unit doseform.

[0146] As used herein, a range is understood to specifically discloseall whole unit positions thereof.

[0147] Typical embodiments of the invention disclosed herein include158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNAsequences (and those which are complementary to such sequences) such asthose that encode the proteins and/or fragments thereof, for example:

[0148] (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 328 ormore than 328 contiguous amino acids of 158P3D2.

[0149] For example, representative embodiments of the inventiondisclosed herein include: polynucleotides and their encoded peptidesthemselves encoding about amino acid 1 to about amino acid 10 of the158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 10 to about amino acid 20 of the 158P3D2 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 158P3D2 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 158P3D2 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 158P3D2 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 158P3D2 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 158P3D2 protein shown in FIG. 2 or FIG. 3, in increments of about 10amino acids, ending at the carboxyl terminal amino acid set forth inFIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of theamino acid sequence (of about 10 amino acids), of amino acids 100through the carboxyl terminal amino acid of the 158P3D2 protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

[0150] Polynucleotides encoding relatively long portions of a 158P3D2protein are also within the scope of the invention. For example,polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 158P3D2protein shown in FIG. 2 or FIG. 3 can be generated by a variety oftechniques well known in the art. These polynucleotide fragments caninclude any portion of the 158P3D2 sequence as shown in FIG. 2.

[0151] Additional illustrative embodiments of the invention disclosedherein include 158P3D2 polynucleotide fragments encoding one or more ofthe biological motifs contained within a 158P3D2 protein sequence,including one or more of the motif-bearing subsequences of a 158P3D2protein set forth in Tables V-XIX. In another embodiment, typicalpolynucleotide fragments of the invention encode one or more of theregions of 158P3D2 protein or variant that exhibit homology to a knownmolecule. In another embodiment of the invention, typical polynucleotidefragments can encode one or more of the 158P3D2 protein or variantN-glycosylation sites, cAMP and cGMP-dependent protein kinasephosphorylation sites, casein kinase II phosphorylation sites orN-myristoylation site and amidation sites.

[0152] II.A.) Uses of 158P3D2 Polynucleotides

[0153] II.A.I.) Monitoring of Genetic Abnormalities

[0154] The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 158P3D2 gene maps to the chromosomallocation set forth in Example 3. For example, because the 158P3D2 genemaps to this chromosome, polynucleotides that encode different regionsof the 158P3D2 proteins are used to characterize cytogeneticabnormalities of this chromosomal locale, such as abnormalities that areidentified as being associated with various cancers. In certain genes, avariety of chromosomal abnormalities including rearrangements have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4):81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) andFinger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotidesencoding specific regions of the 158P3D2 proteins provide new tools thatcan be used to delineate, with greater precision than previouslypossible, cytogenetic abnormalities in the chromosomal region thatencodes 158P3D2 that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

[0155] Furthermore, as 158P3D2 was shown to be highly expressed inbladder and other cancers, 158P3D2 polynucleotides are used in methodsassessing the status of 158P3D2 gene products in normal versus canceroustissues. Typically, polynucleotides that encode specific regions of the158P3D2 proteins are used to assess the presence of perturbations (suchas deletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 158P3D2 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

[0156] II.A.2.) Antisense Embodiments

[0157] Other specifically contemplated nucleic acid related embodimentsof the invention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 158P3D2. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 158P3D2 polynucleotides andpolynucleotide sequences disclosed herein.

[0158] Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 158P3D2 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 158P3D2 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

[0159] The 158P3D2 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a158P3D2 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 158P3D2 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 158P3D2 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 158P3D2 mRNA. Optionally, 158P3D2antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 158P3D2. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 158P3D2 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

[0160] II.A.3.) Primers and Primer Pairs

[0161] Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 158P3D2 polynucleotide in a sample and as ameans for detecting a cell expressing a 158P3D2 protein.

[0162] Examples of such probes include polypeptides comprising all orpart of the human 158P3D2 cDNA sequence shown in FIG. 2. Examples ofprimer pairs capable of specifically amplifying 158P3D2 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 158P3D2 mRNA.

[0163] The 158P3D2 polynucleotides of the invention are useful for avariety of purposes, including but not limited to their use as probesand primers for the amplification and/or detection of the 158P3D2gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosisand/or prognosis of prostate cancer and other cancers; as codingsequences capable of directing the expression of 158P3D2 polypeptides;as tools for modulating or inhibiting the expression of the 158P3D2gene(s) and/or translation of the 158P3D2 transcript(s); and astherapeutic agents.

[0164] The present invention includes the use of any probe as describedherein to identify and isolate a 158P3D2 or 158P3D2 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

[0165] II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid Molecules

[0166] The 158P3D2 cDNA sequences described herein enable the isolationof other polynucleotides encoding 158P3D2 gene product(s), as well asthe isolation of polynucleotides encoding 158P3D2 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a158P3D2 gene product as well as polynucleotides that encode analogs of158P3D2-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding an 158P3D2 gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P3D2gene cDNAs can be identified by probing with a labeled 158P3D2 cDNA or afragment thereof. For example, in one embodiment, a 158P3D2 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 158P3D2gene. A 158P3D2 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 158P3D2 DNAprobes or primers.

[0167] II.A.5.) Recombinant Nucleic Acid Molecules and Host-VectorSystems

[0168] The invention also provides recombinant DNA or RNA moleculescontaining an 158P3D2 polynucleotide, a fragment, analog or homologuethereof, including but not limited to phages, plasmids, phagemids,cosmids, YACs, BACs, as well as various viral and non-viral vectors wellknown in the art, and cells transformed or transfected with suchrecombinant DNA or RNA molecules. Methods for generating such moleculesare well known (see, for example, Sambrook et al., 1989, supra).

[0169] The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 158P3D2 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 158P3D2or a fragment, analog or homolog thereof can be used to generate 158P3D2proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

[0170] A wide range of host-vector systems suitable for the expressionof 158P3D2 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRottkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 158P3D2 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 158P3D2 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 158P3D2 and 158P3D2 mutations or analogs.

[0171] Recombinant human 158P3D2 protein or an analog or homolog orfragment thereof can be produced by mammalian cells transfected with aconstruct encoding a 158P3D2-related nucleotide. For example, 293T cellscan be transfected with an expression plasmid encoding 158P3D2 orfragment, analog or homolog thereof, a 158P3D2-related protein isexpressed in the 293T cells, and the recombinant 158P3D2 protein isisolated using standard purification methods (e.g., affinitypurification using anti-158P3D2 antibodies). In another embodiment, a158P3D2 coding sequence is subcloned into the retroviral vectorpSRαMSVtkneo and used to infect various mammalian cell lines, such asNIH 3T3, TsuPr1, 293 and rat-1 in order to establish 158P3D2 expressingcell lines. Various other expression systems well known in the art canalso be employed. Expression constructs encoding a leader peptide joinedin frame to a 158P3D2 coding sequence can be used for the generation ofa secreted form of recombinant 158P3D2 protein.

[0172] As discussed herein, redundancy in the genetic code permitsvariation in 158P3D2 gene sequences. In particular, it is known in theart that specific host species often have specific codon preferences,and thus one can adapt the disclosed sequence as preferred for a desiredhost. For example, preferred analog codon sequences typically have rarecodons (i.e., codons having a usage frequency of less than about 20% inknown sequences of the desired host) replaced with higher frequencycodons. Codon preferences for a specific species are calculated, forexample, by utilizing codon usage tables available on the INTERNET suchas at URL www.dna.affrc.gojp/˜nakamura/codon.html.

[0173] Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

[0174] II.) 158P3D2-Related Proteins

[0175] Another aspect of the present invention provides 158P3D2-relatedproteins. Specific embodiments of 158P3D2 proteins comprise apolypeptide having all or part of the amino acid sequence of human158P3D2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of158P3D2 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 158P3D2 shown in FIG. 2or FIG. 3.

[0176] In general, naturally occurring allelic variants of human 158P3D2share a high degree of structural identity and homology (e.g., 90% ormore homology). Typically, allelic variants of a 158P3D2 protein containconservative amino acid substitutions within the 158P3D2 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 158P3D2. One class of 158P3D2allelic variants are proteins that share a high degree of homology withat least a small region of a particular 158P3D2 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

[0177] Amino acid abbreviations are provided in Table II. Conservativeamino acid substitutions can frequently be made in a protein withoutaltering either the conformation or the function of the protein.Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 conservative substitutions. Such changes includesubstituting any of isoleucine (I), valine (V), and leucine (L) for anyother of these hydrophobic amino acids; aspartic acid (D) for glutamicacid (E) and vice versa; glutamine (O) for asparagine (N) and viceversa; and serine (S) for threonine (T) and vice versa. Othersubstitutions can also be considered conservative, depending on theenvironment of the particular amino acid and its role in thethree-dimensional structure of the protein. For example, glycine (G) andalanine (A) can frequently be interchangeable, as can alanine (A) andvaline (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, e.g. Table III herein;pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (StanfordUniversity); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al.,J Biol Chem May 19, 1995; 270(20):11882-6).

[0178] Embodiments of the invention disclosed herein include a widevariety of art-accepted variants or analogs of 158P3D2 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.158P3D2 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 158P3D2 variant DNA.

[0179] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

[0180] As defined herein, 158P3D2 variants, analogs or homologs, havethe distinguishing attribute of having at least one epitope that is“cross reactive” with a 158P3D2 protein having an amino acid sequence ofFIG. 3. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 158P3D2 variant alsospecifically binds to a 158P3D2 protein having an amino acid sequenceset forth in FIG. 3. A polypeptide ceases to be a variant of a proteinshown in FIG. 3, when it no longer contains any epitope capable of beingrecognized by an antibody or T cell that specifically binds to thestarting 158P3D2 protein. Those skilled in the art understand thatantibodies that recognize proteins bind to epitopes of varying size, anda grouping of the order of about four or five amino acids, contiguous ornot, is regarded as a typical number of amino acids in a minimalepitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955;Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., JImmunol (1985) 135(4):2598-608.

[0181] Other classes of 158P3D2-related protein variants share 70%, 75%,80%, 85% or 90% or more similarity with an amino acid sequence of FIG.3, or a fragment thereof. Another specific class of 158P3D2 proteinvariants or analogs comprise one or more of the 158P3D2 biologicalmotifs described herein or presently known in the art. Thus, encompassedby the present invention are analogs of 158P3D2 fragments (nucleic oramino acid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

[0182] As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a158P3D2 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a158P3D2 protein shown in FIG. 2 or FIG. 3.

[0183] Moreover, representative embodiments of the invention disclosedherein include polypeptides consisting of about amino acid I to aboutamino acid 10 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 10 to about amino acid 20 ofa 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 20 to about amino acid 30 of a 158P3D2 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 158P3D2 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 158P3D2 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 158P3D2 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a 158P3D2protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

[0184] 158P3D2-related proteins are generated using standard peptidesynthesis technology or using chemical cleavage methods well known inthe art. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a 158P3D2-related protein. In oneembodiment, nucleic acid molecules provide a means to generate definedfragments of a 158P3D2 protein (or variants, homologs or analogsthereof).

[0185] III.A.) Motif-bearing Protein Embodiments

[0186] Additional illustrative embodiments of the invention disclosedherein include 158P3D2 polypeptides comprising the amino acid residuesof one or more of the biological motifs contained within a 158P3D2polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,URL addresses: pfam.wustl.edu/;http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.ac.jp/; www.cbs.dtu.dk/;www.ebi.ac.uk/interpro/scan.html; www.expasy.ch/tools/scnpsit1.html;Epimatrix™ and Epimer™, Brown University,www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

[0187] Motif bearing subsequences of all 158P3D2 variant proteins areset forth and identified in Table XVIII.

[0188] Table XX sets forth several frequently occurring motifs based onpfam searches (see URL address pfam.wustl.edu/). The columns of Table XXlist (1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

[0189] Polypeptides comprising one or more of the 158P3D2 motifsdiscussed above are useful in elucidating the specific characteristicsof a malignant phenotype in view of the observation that the 158P3D2motifs discussed above are associated with growth dysregulation andbecause 158P3D2 is overexpressed in certain cancers (See, e.g., TableI). Casein kinase II, cAMP and camp-dependent protein kinase, andProtein Kinase C, for example, are enzymes known to be associated withthe development of the malignant phenotype (see e.g. Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification also associated withcancer and cancer progression (see e.g., Treston et al., J. Natl. CancerInst. Monogr. (13): 169-175 (1992)).

[0190] In another embodiment, proteins of the invention comprise one ormore of the immunoreactive epitopes identified in accordance withart-accepted methods, such as the peptides set forth in Tables V-XIX.CTL epitopes can be determined using specific algorithms to identifypeptides within an 158P3D2 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, BrownUniversity, URLwww.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptidesthat have sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

[0191] Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

[0192] A variety of references reflect the art regarding theidentification and generation of epitopes in a protein of interest aswell as analogs thereof. See, for example, WO 9733602 to Chesnut et al.;Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol.2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20;Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J.Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6(1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J.Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75(1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum.Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582;O'Sullivanet al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al.,Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 199818(2): 79-92.

[0193] Related embodiments of the inventions include polypeptidescomprising combinations of the different motifs set forth in Table XXI,and/or, one or more of the predicted CTL epitopes of Table V throughTable XIX, and/or, one or more of the T cell binding motifs known in theart. Preferred embodiments contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthe polypeptides. In addition, embodiments which include a number ofeither N-terminal and/or C-terminal amino acid residues on either sideof these motifs may be desirable (to, for example, include a greaterportion of the polypeptide architecture in which the motif is located).Typically the number of N-terminal and/or C-terminal amino acid residueson either side of a motif is between about 1 to about 100 amino acidresidues, preferably 5 to about 50 amino acid residues.

[0194] 158P3D2-related proteins are embodied in many forms, preferablyin isolated form. A purified 158P3D2 protein molecule will besubstantially free of other proteins or molecules that impair thebinding of 158P3D2 to antibody, T cell or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 158P3D2-related proteins include purified158P3D2-related proteins and functional, soluble 158P3D2-relatedproteins. In one embodiment, a functional, soluble 158P3D2 protein orfragment thereof retains the ability to be bound by antibody, T cell orother ligand.

[0195] The invention also provides 158P3D2 proteins comprisingbiologically active fragments of a 158P3D2 amino acid sequence shown inFIG. 2 or FIG. 3. Such proteins exhibit properties of the starting158P3D2 protein, such as the ability to elicit the generation ofantibodies that specifically bind an epitope associated with thestarting 158P3D2 protein; to be bound by such antibodies; to elicit theactivation of HTL or CTL; and/or, to be recognized by HTL or CTL thatalso specifically bind to the starting protein.

[0196] 158P3D2-related polypeptides that contain particularlyinteresting structures can be predicted and/or identified using variousanalytical techniques well known in the art, including, for example, themethods of Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis, or on the basis ofimmunogenicity. Fragments that contain such structures are particularlyuseful in generating subunit-specific anti-158P3D2 antibodies, or Tcells or in identifying cellular factors that bind to 158P3D2. Forexample, hydrophilicity profiles can be generated, and immunogenicpeptide fragments identified, using the method of Hopp, T. P. and Woods,K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

[0197] CTL epitopes can be determined using specific algorithms toidentify peptides within an 158P3D2 protein that are capable ofoptimally binding to specified HLA alleles (e.g., by using the SYFPEITHIsite at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listingsin Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL(www.brown.edu/Research/TB-HIV Lab/epimatrix/epimatrix.html); and BIMAS,URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from158P3D2 that are presented in the context of human MHC class I moleculesHLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XIX).Specifically, the complete amino acid sequence of the 158P3D2 proteinand relevant portions of other variants, i.e., for HLA Class Ipredictions 9 flanking residues on either side of a point mutation, andfor HLA Class II predictions 14 flanking residues on either side of apoint mutation, were entered into the HLA Peptide Motif Search algorithmfound in the Bioinformatics and Molecular Analysis Section (BIMAS) website listed above; for HLA Class II the site SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/ was used.

[0198] The HLA peptide motif search algorithm was developed by Dr. KenParker based on binding of specific peptide sequences in the groove ofHLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al.,Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parkeret al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 158P3D2 predicted binding peptides areshown in Tables V-XIX herein. In Tables V-XIX, the top rankingcandidates, 9-mers, 10-mers and 15-mers (for each family member), areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

[0199] Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

[0200] It is to be appreciated that every epitope predicted by the BIMASsite, Epimer™ and Epimatrix™ sites, or specified by the HLA class I orclass II motifs available in the art or which become part of the artsuch as set forth in Table IV (or determined using World Wide Web siteURL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are tobe “applied” to a 158P3D2 protein in accordance with the invention. Asused in this context “applied” means that a 158P3D2 protein isevaluated, e.g., visually or by computer-based patterns finding methods,as appreciated by those of skill in the relevant art. Every subsequenceof a 158P3D2 protein of 8, 9, 10, or II amino acid residues that bearsan HLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

[0201] III.B.) Expression of 158P3D2-Related Proteins

[0202] In an embodiment described in the examples that follow, 158P3D2can be conveniently expressed in cells (such as 293T cells) transfectedwith a commercially available expression vector such as a CMV-drivenexpression vector encoding 158P3D2 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 158P3D2 protein intransfected cells. The secreted HIS-tagged 158P3D2 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

[0203] III.C.) Modifications of 158P3D2-Related Proteins

[0204] Modifications of 158P3D2-related proteins such as covalentmodifications are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a 158P3D2 polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a 158P3D2 protein. Another type of covalent modification ofa 158P3D2 polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of a protein of theinvention. Another type of covalent modification of 158P3D2 compriseslinking a 158P3D2 polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0205] The 158P3D2-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 158P3D2 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a158P3D2 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 158P3D2. Achimeric molecule can comprise a fusion of a 158P3D2-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a 158P3D2 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a 158P3D2-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 158P3D2polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgG1 molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

[0206] III.D.) Uses of 158P3D2-Related Proteins

[0207] The proteins of the invention have a number of different specificuses. As 158P3D2 is highly expressed in prostate and other cancers,158P3D2-related proteins are used in methods that assess the status of158P3D2 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 158P3D2 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 158P3D2-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 1 58P3D2 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,158P3D2-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 158P3D2 protein are used to screenfor factors that interact with that region of 158P3D2.

[0208] 158P3D2 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an158P3D2 protein), for identifying agents or cellular factors that bindto 158P3D2 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

[0209] Proteins encoded by the 158P3D2 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 158P3D2 gene productAntibodies raised against an 158P3D2 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 158P3D2protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 158P3D2-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

[0210] Various immunological assays useful for the detection of 158P3D2proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 158P3D2-expressingcells (e.g., in radioscintigraphic imaging methods). 158P3D2 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

[0211] IV.) 158P3D2 Antibodies

[0212] Another aspect of the invention provides antibodies that bind to158P3D2-related proteins. Preferred antibodies specifically bind to a158P3D2-related protein and do not bind (or bind weakly) to peptides orproteins that are not 158P3D2-related proteins. For example, antibodiesthat bind 158P3D2 can bind 158P3D2-related proteins such as the homologsor analogs thereof.

[0213] 158P3D2 antibodies of the invention are particularly useful incancer (see, e.g., Table I) diagnostic and prognostic assays, andimaging methodologies. Similarly, such antibodies are useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extent158P3D2 is also expressed or overexpressed in these other cancers.Moreover, intracellularly expressed antibodies (e.g., single chainantibodies) are therapeutically useful in treating cancers in which theexpression of 158P3D2 is involved, such as advanced or metastaticprostate cancers.

[0214] The invention also provides various immunological assays usefulfor the detection and quantification of 158P3D2 and mutant158P3D2-related proteins. Such assays can comprise one or more 158P3D2antibodies capable of recognizing and binding a 158P3D2-related protein,as appropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

[0215] Immunological non-antibody assays of the invention also compriseT cell immunogenicity assays (inhibitory or stimulatory) as well asmajor histocompatibility complex (MHC) binding assays.

[0216] In addition, immunological imaging methods capable of detectingprostate cancer and other cancers expressing 158P3D2 are also providedby the invention, including but not limited to radioscintigraphicimaging methods using labeled 158P3D2 antibodies. Such assays areclinically useful in the detection, monitoring, and prognosis of 158P3D2expressing cancers such as prostate cancer.

[0217] 158P3D2 antibodies are also used in methods for purifying a158P3D2-related protein and for isolating 158P3D2 homologues and relatedmolecules. For example, a method of purifying a 158P3D2-related proteincomprises incubating an 158P3D2 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a158P3D2-related protein under conditions that permit the 158P3D2antibody to bind to the 158P3D2-related protein; washing the solidmatrix to eliminate impurities; and eluting the 158P3D2-related proteinfrom the coupled antibody. Other uses of 158P3D2 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 158P3D2 protein.

[0218] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies can be prepared by immunizing asuitable mammalian host using a 158P3D2-related protein, peptide, orfragment, in isolated or immunoconjugated form (Antibodies: A LaboratoryManual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies,Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of158P3D2 can also be used, such as a 158P3D2 GST-fusion protein. In aparticular embodiment, a GST fusion protein comprising all or most ofthe amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as animmunogen to generate appropriate antibodies. In another embodiment, a158P3D2-related protein is synthesized and used as an immunogen.

[0219] In addition, naked DNA immunization techniques known in the artare used (with or without purified 158P3D2-related protein or 158P3D2expressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0220] The amino acid sequence of a 158P3D2 protein as shown in FIG. 2or FIG. 3 can be analyzed to select specific regions of the 158P3D2protein for generating antibodies. For example, hydrophobicity andhydrophilicity analyses of a 158P3D2 amino acid sequence are used toidentify hydrophilic regions in the 158P3D2 structure. Regions of a158P3D2 protein that show immunogenic structure, as well as otherregions and domains, can readily be identified using various othermethods known in the art, such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Hydrophilicity profiles can be generated using the method of Hopp, T. P.and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828.Hydropathicity profiles can be generated using the method of Kyte, J.and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%)Accessible Residues profiles can be generated using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int.J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generatedusing the method of Deleage, G., Roux B., 1987, Protein Engineering1:289-294. Thus, each region identified by any of these programs ormethods is within the scope of the present invention. Methods for thegeneration of 158P3D2 antibodies are further illustrated by way of theexamples provided herein. Methods for preparing a protein or polypeptidefor use as an immunogen are well known in the art. Also well known inthe art are methods for preparing immunogenic conjugates of a proteinwith a carrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 158P3D2 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

[0221] 158P3D2 monoclonal antibodies can be produced by various meanswell known in the art. For example, immortalized cell lines that secretea desired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 158P3D2-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

[0222] The antibodies or fragments of the invention can also beproduced, by recombinant means. Regions that bind specifically to thedesired regions of a 158P3D2 protein can also be produced in the contextof chimeric or complementarity determining region (CDR) graftedantibodies of multiple species origin. Humanized or human 158P3D2antibodies can also be produced, and are preferred for use intherapeutic contexts. Methods for humanizing murine and other non-humanantibodies, by substituting one or more of the non-human antibody CDRsfor corresponding human antibody sequences, are well known (see forexample, Jones et al., 1986, Nature 321: 522-525; Riechmann et a., 1988,Nature 332: 323-327; Verhoeyen et at, 1988, Science 239: 1534-1536). Seealso, Carter et at, 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Simset at, 1993, J. Immunol. 151: 2296.

[0223] Methods for producing fully human monoclonal antibodies includephage display and transgenic methods (for review, see Vaughan et a.,1998, Nature Biotechnology 16: 535-539). Fully human 158P3D2 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human158P3D2 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO 98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued Dec. 19, 2000; U.S.Pat. No. 6,150,584 issued Nov. 12, 2000; and, U.S. Pat. No. 6,114,598issued Sep. 5, 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

[0224] Reactivity of 158P3D2 antibodies with an 158P3D2-related proteincan be established by a number of well known means, including Westernblot, immunoprecipitation, ELISA, and FACS analyses using, asappropriate, 158P3D2-related proteins, 158P3D2-expressing cells orextracts thereof. A 158P3D2 antibody or fragment thereof can be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 158P3D2 epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al, CancerRes. 53: 2560-2565).

[0225] V.) 158P3D2 Cellular Immune Responses

[0226] The mechanism by which T cells recognize antigens has beendelineated. Efficacious peptide epitope vaccine compositions of theinvention induce a therapeutic or prophylactic immune responses in verybroad segments of the world-wide population. For an understanding of thevalue and efficacy of compositions of the invention that induce cellularimmune responses, a brief review of immunology-related technology isprovided.

[0227] A complex of an HLA molecule and a peptidic antigen acts as theligand recognized by HLA-restricted T cells (Buus, S. et al., Cell47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A.and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu.Rev. Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, eta., J. Immunol. 160:3363, 1998; Rammensee, et a., Immunogenetics 41:178,1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URLsyfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al.,Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995;Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., HumanImmunol. 45:79-93, 1996; Sette, A. and Sidney, J. ImmunogeneticsNovember 1999; 50(3-4):201-12, Review).

[0228] Furthermore, x-ray crystallographic analyses of HLA-peptidecomplexes have revealed pockets within the peptide binding cleft/grooveof HLA molecules which accommodate, in an allele-specific mode, residuesborne by peptide ligands; these residues in turn determine the HLAbinding capacity of the peptides in which they are present. (See, e.g.,Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stem et al.,Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997;Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl.Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992;Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al.,Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H.et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley,D. C., J. Mol. Biol. 219:277, 1991.)

[0229] Accordingly, the definition of class I and class IIallele-specific HLA binding motifs, or class I or class II supermotifsallows identification of regions within a protein that are correlatedwith binding to particular HLA antigen(s).

[0230] Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

[0231] Various strategies can be utilized to evaluate cellularimmunogenicity, including:

[0232] 1) Evaluation of primary T cell cultures from normal individuals(see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis,E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

[0233] 2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P.A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int.Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997).For example, in such methods peptides in incomplete Freund's adjuvantare administered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

[0234] 3) Demonstration of recall T cell responses from immuneindividuals who have been either effectively vaccinated and/or fromchronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med.181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R.et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J.Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011,1997). Accordingly, recall responses are detected by culturing PBL fromsubjects that have been exposed to the antigen due to disease and thushave generated an immune response “naturally”, or from patients who werevaccinated against the antigen. PBL from subjects are cultured in vitrofor 1-2 weeks in the presence of test peptide plus antigen presentingcells (APC) to allow activation of “memory” T cells, as compared to“naive” T cells. At the end of the culture period, T cell activity isdetected using assays including ⁵¹Cr release involvingpeptide-sensitized targets, T cell proliferation, or lymphokine release.

[0235] VI.) 158P3D2 Transgenic Animals

[0236] Nucleic acids that encode a 158P3D2-related protein can also beused to generate either transgenic animals or “knock out” animals that,in turn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 158P3D2 can be used to clone genomic DNA that encodes 158P3D2.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 158P3D2. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued Apr. 12, 1988, and U.S. Pat.No. 4,870,009 issued Sep. 26, 1989. Typically, particular cells would betargeted for 158P3D2 transgene incorporation with tissue-specificenhancers.

[0237] Transgenic animals that include a copy of a transgene encoding158P3D2 can be used to examine the effect of increased expression of DNAthat encodes 158P3D2. Such animals can be used as tester animals forreagents thought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with thisaspect of the invention, an animal is treated with a reagent and areduced incidence of a pathological condition, compared to untreatedanimals that bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

[0238] Alternatively, non-human homologues of 158P3D2 can be used toconstruct a 158P3D2 “knock out” animal that has a defective or alteredgene encoding 158P3D2 as a result of homologous recombination betweenthe endogenous gene encoding 158P3D2 and altered genomic DNA encoding158P3D2 introduced into an embryonic cell of the animal. For example,cDNA that encodes 158P3D2 can be used to clone genomic DNA encoding158P3D2 in accordance with established techniques. A portion of thegenomic DNA encoding 158P3D2 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 158P3D2 polypeptide.

[0239] VII.) Methods for the Detection of 158P3D2

[0240] Another aspect of the present invention relates to methods fordetecting 158P3D2 polynucleotides and 158P3D2-related proteins, as wellas methods for identifying a cell that expresses 158P3D2. The expressionprofile of 158P3D2 makes it a diagnostic marker for metastasizeddisease. Accordingly, the status of 158P3D2 gene products providesinformation useful for predicting a variety of factors includingsusceptibility to advanced stage disease, rate of progression, and/ortumor aggressiveness. As discussed in detail herein, the status of158P3D2 gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

[0241] More particularly, the invention provides assays for thedetection of 158P3D2 polynucleotides in a biological sample, such asserum, bone, prostate, and other tissues, urine, semen, cellpreparations, and the like. Detectable 158P3D2 polynucleotides include,for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA,alternative splice variant 158P3D2 mRNAs, and recombinant DNA or RNAmolecules that contain a 158P3D2 polynucleotide. A number of methods foramplifying and/or detecting the presence of 158P3D2 polynucleotides arewell known in the art and can be employed in the practice of this aspectof the invention.

[0242] In one embodiment, a method for detecting an 158P3D2 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 158P3D2 polynucleotides as sense and antisense primers toamplify 158P3D2 cDNAs therein; and detecting the presence of theamplified 158P3D2 cDNA. Optionally, the sequence of the amplified158P3D2 cDNA can be determined.

[0243] In another embodiment, a method of detecting a 158P3D2 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 158P3D2 polynucleotides assense and antisense primers; and detecting the presence of the amplified158P3D2 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 158P3D2 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

[0244] The invention also provides assays for detecting the presence ofan 158P3D2 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 158P3D2-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 158P3D2-related proteinin a biological sample comprises first contacting the sample with a158P3D2 antibody, a 158P3D2-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 158P3D2 antibody; andthen detecting the binding of 158P3D2-related protein in the sample.

[0245] Methods for identifying a cell that expresses 158P3D2 are alsowithin the scope of the invention. In one embodiment, an assay foridentifying a cell that expresses a 158P3D2 gene comprises detecting thepresence of 158P3D2 mRNA in the cell. Methods for the detection ofparticular mRNAs in cells are well known and include, for example,hybridization assays using complementary DNA probes (such as in situhybridization using labeled 158P3D2 riboprobes, Northern blot andrelated techniques) and various nucleic acid amplification assays (suchas RT-PCR using complementary primers specific for 158P3D2, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like). Alternatively, an assay for identifying acell that expresses a 158P3D2 gene comprises detecting the presence of158P3D2-related protein in the cell or secreted by the cell. Variousmethods for the detection of proteins are well known in the art and areemployed for the detection of 158P3D2-related proteins and cells thatexpress 158P3D2-related proteins.

[0246] 158P3D2 expression analysis is also useful as a tool foridentifying and evaluating agents that modulate 158P3D2 gene expression.For example, 158P3D2 expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 158P3D2expression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 158P3D2 expression by RT-PCR, nucleic acid hybridization orantibody binding.

[0247] VIII.) Methods for Monitoring the Status of 158P3D2-Related Genesand Their Products

[0248] Oncogenesis is known to be a multistep process where cellulargrowth becomes progressively dysregulated and cells progress from anormal physiological state to precancerous and then cancerous states(see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacset al., Cancer Surv. 23: 19-32 (1995)). In this context, examining abiological sample for evidence of dysregulated cell growth (such asaberrant 158P3D2 expression in cancers) allows for early detection ofsuch aberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 158P3D2 in abiological sample of interest can be compared, for example, to thestatus of 158P3D2 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 158P3D2 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare158P3D2 status in a sample.

[0249] The term “status” in this context is used according to its artaccepted meaning and refers to the condition or state of a gene and itsproducts. Typically, skilled artisans use a number of parameters toevaluate the condition or state of a gene and its products. Theseinclude, but are not limited to the location of expressed gene products(including the location of 158P3D2 expressing cells) as well as thelevel, and biological activity of expressed gene products (such as158P3D2 mRNA, polynucleotides and polypeptides). Typically, analteration in the status of 158P3D2 comprises a change in the locationof 158P3D2 and/or 158P3D2 expressing cells and/or an increase in 158P3D2mRNA and/or protein expression.

[0250] 158P3D2 status in a sample can be analyzed by a number of meanswell known in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 158P3D2 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 158P3D2 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 158P3D2 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 158P3D2 proteins and/orassociations of 158P3D2 proteins with polypeptide binding partners).Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 geneor fragment thereof, 158P3D2 mRNA, alternative splice variants, 158P3D2mRNAs, and recombinant DNA or RNA molecules containing a 158P3D2polynucleotide.

[0251] The expression profile of 158P3D2 makes it a diagnostic markerfor local and/or metastasized disease, and provides information on thegrowth or oncogenic potential of a biological sample. In particular, thestatus of 158P3D2 provides information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining 158P3D2 status and diagnosing cancers that express 158P3D2,such as cancers of the tissues listed in Table I. For example, because158P3D2 mRNA is so highly expressed in prostate and other cancersrelative to normal prostate tissue, assays that evaluate the levels of158P3D2 mRNA transcripts or proteins in a biological sample can be usedto diagnose a disease associated with 158P3D2 dysregulation, and canprovide prognostic information useful in defining appropriatetherapeutic options.

[0252] The expression status of 158P3D2 provides information includingthe presence, stage and location of dysplastic, precancerous andcancerous cells, predicting susceptibility to various stages of disease,and/or for gauging tumor aggressiveness. Moreover, the expressionprofile makes it useful as an imaging reagent for metastasized disease.Consequently, an aspect of the invention is directed to the variousmolecular prognostic and diagnostic methods for examining the status of158P3D2 in biological samples such as those from individuals sufferingfrom, or suspected of suffering from a pathology characterized bydysregulated cellular growth, such as cancer.

[0253] As described above, the status of 158P3D2 in a biological samplecan be examined by a number of well-known procedures in the art. Forexample, the status of 158P3D2 in a biological sample taken from aspecific location in the body can be examined by evaluating the samplefor the presence or absence of 158P3D2 expressing cells (e.g. those thatexpress 158P3D2 mRNAs or proteins). This examination can provideevidence of dysregulated cellular growth, for example, when158P3D2-expressing cells are found in a biological sample that does notnormally contain such cells (such as a lymph node), because suchalterations in the status of 158P3D2 in a biological sample are oftenassociated with dysregulated cellular growth. Specifically, oneindicator of dysregulated cellular growth is the metastases of cancercells from an organ of origin (such as the prostate) to a different areaof the body (such as a lymph node). In this context, evidence ofdysregulated cellular growth is important for example because occultlymph node metastases can be detected in a substantial proportion ofpatients with prostate cancer, and such metastases are associated withknown predictors of disease progression (see, e.g., Murphy et al.,Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1):17-28 (2000) and Freeman et al., J Urol August 1995 154(2 Pt 1):474-8).

[0254] In one aspect, the invention provides methods for monitoring158P3D2 gene products by determining the status of 158P3D2 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 158P3D2gene products in a corresponding normal sample. The presence of aberrant158P3D2 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

[0255] In another aspect, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant increase in 158P3D2 mRNA or protein expressionin a test cell or tissue sample relative to expression levels in thecorresponding normal cell or tissue. The presence of 158P3D2 mRNA can,for example, be evaluated in tissue samples including but not limited tothose listed in Table I. The presence of significant 158P3D2 expressionin any of these tissues is useful to indicate the emergence, presenceand/or severity of a cancer, since the corresponding normal tissues donot express 158P3D2 mRNA or express it at lower levels.

[0256] In a related embodiment, 158P3D2 status is determined at theprotein level rather than at the nucleic acid level. For example, such amethod comprises determining the level of 158P3D2 protein expressed bycells in a test tissue sample and comparing the level so determined tothe level of 158P3D2 expressed in a corresponding normal sample. In oneembodiment, the presence of 158P3D2 protein is evaluated, for example,using immunohistochemical methods. 158P3D2 antibodies or bindingpartners capable of detecting 158P3D2 protein expression are used in avariety of assay formats well known in the art for this purpose.

[0257] In a further embodiment, one can evaluate the status of 158P3D2nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 158P3D2 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 158P3D2 indicates a potential lossof function or increase in tumor growth.

[0258] A wide variety of assays for observing perturbations innucleotide and amino acid sequences are well known in the art. Forexample, the size and structure of nucleic acid or amino acid sequencesof 158P3D2 gene products are observed by the Northern, Southern,Western, PCR and DNA sequencing protocols discussed herein. In addition,other methods for observing perturbations in nucleotide and amino acidsequences such as single strand conformation polymorphism analysis arewell known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued Sep. 7,1999, and U.S. Pat. No. 5,952,170 issued Jan. 17, 1995).

[0259] Additionally, one can examine the methylation status of a 158P3D2gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in>90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-I tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). A variety of assays for examining methylation status ofa gene are well known in the art. For example, one can utilize, inSouthern hybridization approaches, methylation-sensitive restrictionenzymes that cannot cleave sequences that contain methylated CpG sitesto assess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubel et al. eds., 1995.

[0260] Gene amplification is an additional method for assessing thestatus of 158P3D2. Gene amplification is measured in a sample directly,for example, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

[0261] Biopsied tissue or peripheral blood can be conveniently assayedfor the presence of cancer cells using for example, Northern, dot blotor RT-PCR analysis to detect 158P3D2 expression. The presence of RT-PCRamplifiable 158P3D2 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

[0262] A further aspect of the invention is an assessment of thesusceptibility that an individual has for developing cancer. In oneembodiment, a method for predicting susceptibility to cancer comprisesdetecting 158P3D2 mRNA or 158P3D2 protein in a tissue sample, itspresence indicating susceptibility to cancer, wherein the degree of158P3D2 mRNA expression correlates to the degree of susceptibility. In aspecific embodiment, the presence of 158P3D2 in prostate or other tissueis examined, with the presence of 158P3D2 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 158P3D2 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 158P3D2 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

[0263] The invention also comprises methods for gauging tumoraggressiveness. In one embodiment, a method for gauging aggressivenessof a tumor comprises determining the level of 158P3D2 mRNA or 158P3D2protein expressed by tumor cells, comparing the level so determined tothe level of 158P3D2 mRNA or 158P3D2 protein expressed in acorresponding normal tissue taken from the same individual or a normaltissue reference sample, wherein the degree of 158P3D2 mRNA or 158P3D2protein expression in the tumor sample relative to the normal sampleindicates the degree of aggressiveness. In a specific embodiment,aggressiveness of a tumor is evaluated by determining the extent towhich 158P3D2 is expressed in the tumor cells, with higher expressionlevels indicating more aggressive tumors. Another embodiment is theevaluation of the integrity of 158P3D2 nucleotide and amino acidsequences in a biological sample, in order to identify perturbations inthe structure of these molecules such as insertions, deletions,substitutions and the like. The presence of one or more perturbationsindicates more aggressive tumors.

[0264] Another embodiment of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 158P3D2 mRNAor 158P3D2 protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of 158P3D2 mRNA or158P3D2 protein expressed in an equivalent tissue sample taken from thesame individual at a different time, wherein the degree of 158P3D2 mRNAor 158P3D2 protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining 158P3D2expression in the tumor cells over time, where increased expression overtime indicates a progression of the cancer. Also, one can evaluate theintegrity 158P3D2 nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,where the presence of one or more perturbations indicates a progressionof the cancer.

[0265] The above diagnostic approaches can be combined with any one of awide variety of prognostic and diagnostic protocols known in the art.For example, another embodiment of the invention is directed to methodsfor observing a coincidence between the expression of 158P3D2 gene and158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-5 1; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 158P3D2 gene and 158P3D2 gene products (or perturbationsin 158P3D2 gene and 158P3D2 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

[0266] In one embodiment, methods for observing a coincidence betweenthe expression of 158P3D2 gene and 158P3D2 gene products (orperturbations in 158P3D2 gene and 158P3D2 gene products) and anotherfactor associated with malignancy entails detecting the overexpressionof 158P3D2 mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSMexpression), and observing a coincidence of 158P3D2 mRNA or protein andPSA mRNA or protein overexpression (or PSCA or PSM expression). In aspecific embodiment, the expression of 158P3D2 and PSA mRNA in prostatetissue is examined, where the coincidence of 158P3D2 and PSA mRNAoverexpression in the sample indicates the existence of prostate cancer,prostate cancer susceptibility or the emergence or status of a prostatetumor.

[0267] Methods for detecting and quantifying the expression of 158P3D2mRNA or protein are described herein, and standard nucleic acid andprotein detection and quantification technologies are well known in theart. Standard methods for the detection and quantification of 158P3D2mRNA include in situ hybridization using labeled 158P3D2 riboprobes,Northern blot and related techniques using 158P3D2 polynucleotideprobes, RT-PCR analysis using primers specific for 158P3D2, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR is used to detect and quantify 158P3D2 mRNAexpression. Any number of primers capable of amplifying 158P3D2 can beused for this purpose, including but not limited to the various primersets specifically described herein. In a specific embodiment, polyclonalor monoclonal antibodies specifically reactive with the wild-type158P3D2 protein can be used in an immunohistochemical assay of biopsiedtissue.

[0268] IX.) Identification of Molecules that Interact with 158P3D2

[0269] The 158P3D2 protein and nucleic acid sequences disclosed hereinallow a skilled artisan to identify proteins, small molecules and otheragents that interact with 158P3D2, as well as pathways activated by-158P3D2 via any one of a variety of art accepted protocols. Forexample, one can utilize one of the so-called interaction trap systems(also referred to as the “two-hybrid assay”). In such systems, moleculesinteract and reconstitute a transcription factor which directsexpression of a reporter gene, whereupon the expression of the reportergene is assayed. Other systems identify protein-protein interactions invivo through reconstitution of a eukaryotic transcriptional activator,see, e.g., U.S. Pat. No. 5,955,280 issued Sep. 21, 1999, U.S. Pat. No.5,925,523 issued Jul. 20, 1999, U.S. Pat. No. 5,846,722 issued Dec. 8,1998 and U.S. Pat. No. 6,004,746 issued Dec. 21, 1999. Algorithms arealso available in the art for genome-based predictions of proteinfunction (see, e.g., Marcotte, et al., Nature 402: Nov. 4, 1999, 83-86).

[0270] Alternatively one can screen peptide libraries to identifymolecules that interact with 158P3D2 protein sequences. In such methods,peptides that bind to 158P3D2 are identified by screening libraries thatencode a random or controlled collection of amino acids. Peptidesencoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the 158P3D2 protein(s).

[0271] Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 158P3D2 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issuedMar. 3, 1998 and U.S. Pat. No. 5,733,731 issued Mar. 31, 1998.

[0272] Alternatively, cell lines that express 158P3D2 are used toidentify protein-protein interactions mediated by 158P3D2. Suchinteractions can be examined using immunoprecipitation techniques (see,e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999,261:646-51). 158P3D2 protein can be immunoprecipitated from158P3D2-expressing cell lines using anti-158P3D2 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express fusions of 158P3D2 and a His-tag (vectorsmentioned above). The immunoprecipitated complex can be examined forprotein association by procedures such as Western blotting,³⁵S-methionine labeling of proteins, protein microsequencing, silverstaining and two-dimensional gel electrophoresis.

[0273] Small molecules and ligands that interact with 158P3D2 can beidentified through related embodiments of such screening assays. Forexample, small molecules can be identified that interfere with proteinfunction, including molecules that interfere with 158P3D2's ability tomediate phosphorylation and de-phosphorylation, interaction with DNA orRNA molecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 158P3D2-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 158P3D2 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 158P3D2 function can beidentified based on their ability to bind 158P3D2 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of158P3D2 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 158P3D2.

[0274] An embodiment of this invention comprises a method of screeningfor a molecule that interacts with an 158P3D2 amino acid sequence shownin FIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 158P3D2 amino acid sequence, allowing the population ofmolecules and the 158P3D2 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 158P3D2 amino acid sequence, and thenseparating molecules that do not interact with the 158P3D2 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 158P3D2 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 158P3D2. In apreferred embodiment, the 158P3D2 amino acid sequence is contacted witha library of peptides.

[0275] X.) Therapeutic Methods and Compositions

[0276] The identification of 158P3D2 as a protein that is normallyexpressed in a restricted set of tissues, but which is also expressed inprostate and other cancers, opens a number of therapeutic approaches tothe treatment of such cancers. As contemplated herein, 158P3D2 functionsas a transcription factor involved in activating tumor-promoting genesor repressing genes that block tumorigenesis.

[0277] Accordingly, therapeutic approaches that inhibit the activity ofa 158P3D2 protein are useful for patients suffering from a cancer thatexpresses 158P3D2. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 158P3D2 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 158P3D2 gene or translation of 158P3D2mRNA.

[0278] X.A.) Anti-Cancer Vaccines

[0279] The invention provides cancer vaccines comprising a158P3D2-related protein or 158P3D2-related nucleic acid. In view of theexpression of 158P3D2, cancer vaccines prevent and/or treat158P3D2-expressing cancers with minimal or no effects on non-targettissues. The use of a tumor antigen in a vaccine that generates humoraland/or cell-mediated immune responses as anti-cancer therapy is wellknown in the art and has been employed in prostate cancer using humanPSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

[0280] Such methods can be readily practiced by employing a158P3D2-related protein, or an 158P3D2-encoding nucleic acid moleculeand recombinant vectors capable of expressing and presenting the 158P3D2immunogen (which typically comprises a number of antibody or T cellepitopes). Skilled artisans understand that a wide variety of vaccinesystems for delivery of immunoreactive epitopes are known in the art(see, e.g., Heryln et al., Ann Med February 1999 31(1):66-78; Maruyamaet al., Cancer Immunol Immunother June 2000 49(3): 123-32) Briefly, suchmethods of generating an immune response (e.g. humoral and/orcell-mediated) in a mammal, comprise the steps of: exposing the mammal'simmune system to an immunoreactive epitope (e.g. an epitope present in a158P3D2 protein shown in FIG. 3 or analog or homolog thereof) so thatthe mammal generates an immune response that is specific for thatepitope (e.g. generates antibodies that specifically recognize thatepitope). In a preferred method, a 158P3D2 immunogen contains abiological motif, see e.g., Tables V-XIX, or a peptide of a size rangefrom 158P3D2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

[0281] The entire 158P3D2 protein, immunogenic regions or epitopesthereof can be combined and delivered by various means. Such vaccinecompositions can include, for example, lipopeptides (e.g., Vitiello, A.et al., J. Clin. Invest. 95:341, 1995), peptide compositionsencapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see,e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),peptide compositions contained in immune stimulating complexes (ISCOMS)(see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., ClinExp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M. -P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

[0282] In patients with 158P3D2-associated cancer, the vaccinecompositions of the invention can also be used in conjunction with othertreatments used for cancer, e.g., surgery, chemotherapy, drug therapies,radiation therapies, etc. including use in combination with immuneadjuvants such as IL-2, IL-12, GM-CSF, and the like.

[0283] Cellular Vaccines:

[0284] CTL epitopes can be determined using specific algorithms toidentify peptides within 158P3D2 protein that bind corresponding HLAalleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University(URL www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and,BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 158P3D2immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesV-XIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLAClass I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV(E)) and/or a peptide of at least 9 amino acids that comprises an HLAClass II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As isappreciated in the art, the HLA Class I binding groove is essentiallyclosed ended so that peptides of only a particular size range can fitinto the groove and be bound, generally HLA Class I epitopes are 8, 9,10, or 11 amino acids long. In contrast, the HLA Class II binding grooveis essentially open ended; therefore a peptide of about 9 or more aminoacids can be bound by an HLA Class II molecule. Due to the bindinggroove differences between HLA Class I and II, HLA Class I motifs arelength specific, i.e., position two of a Class I motif is the secondamino acid in an amino to carboxyl direction of the peptide. The aminoacid positions in a Class II motif are relative only to each other, notthe overall peptide, i.e., additional amino acids can be attached to theamino and/or carboxyl termini of a motif-bearing sequence. HLA Class IIepitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

[0285] Antibody-Based Vaccines

[0286] A wide variety of methods for generating an immune response in amammal are known in the art (for example as the first step in thegeneration of hybridomas). Methods of generating an immune response in amammal comprise exposing the mammal's immune system to an immunogenicepitope on a protein (e.g. a 158P3D2 protein) so that an immune responseis generated. A typical embodiment consists of a method for generatingan immune response to 158P3D2 in a host, by contacting the host with asufficient amount of at least one 158P3D2 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 158P3D2 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 158P3D2-related protein or aman-made multiepitopic peptide comprising: administering 158P3D2immunogen (e.g. a 158P3D2 protein or a peptide fragment thereof, an158P3D2 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 158P3D2 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 158P3D2 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 158P3D2, in order to generate a response to thetarget antigen.

[0287] Nucleic Acid Vaccines:

[0288] Vaccine compositions of the invention include nucleicacid-mediated modalities. DNA or RNA that encode protein(s) of theinvention can be administered to a patient. Genetic immunization methodscan be employed to generate prophylactic or therapeutic humoral andcellular immune responses directed against cancer cells expressing158P3D2. Constructs comprising DNA encoding a 158P3D2-relatedprotein/immunogen and appropriate regulatory sequences can be injecteddirectly into muscle or skin of an individual, such that the cells ofthe muscle or skin take-up the construct and express the encoded 158P3D2protein/immunogen. Alternatively, a vaccine comprises a 158P3D2-relatedprotein. Expression of the 158P3D2-related protein immunogen results inthe generation of prophylactic or therapeutic humoral and cellularimmunity against cells that bear a 158P3D2 protein. Various prophylacticand therapeutic genetic immunization techniques known in the art can beused (for review, see information and references published at Internetaddress www.genweb.com). Nucleic acid-based delivery is described, forinstance, in Wolff et. al, Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

[0289] For therapeutic or prophylactic immunization purposes, proteinsof the invention can be expressed via viral or bacterial vectors.Various viral gene delivery systems that can be used in the practice ofthe invention include, but are not limited to, vaccinia, fowlpox,canarypox, adenovirus, influenza, poliovirus, adeno-associated virus,lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin.Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990(1995)). Non-viral delivery systems can also be employed by introducingnaked DNA encoding a 158P3D2-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

[0290] Vaccinia virus is used, for example, as a vector to expressnucleotide sequences that encode the peptides of the invention. Uponintroduction into a host, the recombinant vaccinia virus expresses theprotein immunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

[0291] Thus, gene delivery systems are used to deliver a 158P3D2-relatednucleic acid molecule. In one embodiment, the full-length human 158P3D2cDNA is employed. In another embodiment, 158P3D2 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

[0292] Ex Vivo Vaccines

[0293] Various ex vivo strategies can also be employed to generate animmune response. One approach involves the use of antigen presentingcells (APCS) such as dendritic cells (DC) to present 158P3D2 antigen toa patient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 158P3D2 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with158P3D2 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 158P3D2 protein. Yet another embodiment involves engineeringthe overexpression of a 158P3D2 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 158P3D2 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

[0294] X.B.) 158P3D2 as a Target for Antibody-Based Therapy

[0295] 158P3D2 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 158P3D2 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of158P3D2-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 158P3D2 areuseful to treat 158P3D2-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

[0296] 158P3D2 antibodies can be introduced into a patient such that theantibody binds to 158P3D2 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 158P3D2,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

[0297] Those skilled in the art understand that antibodies can be usedto specifically target and bind immunogenic molecules such as animmunogenic region of a 158P3D2 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 158P3D2), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

[0298] A wide variety of compositions and methods for usingantibody-cytotoxic agent conjugates to kill cells are known in the art.In the context of cancers, typical methods entail administering to ananimal having a tumor a biologically effective amount of a conjugatecomprising a selected cytotoxic and/or therapeutic agent linked to atargeting agent (e.g. an anti-158P3D2 antibody) that binds to a marker(e.g. 158P3D2) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment is a method of delivering a cytotoxicand/or therapeutic agent to a cell expressing 158P3D2, comprisingconjugating the cytotoxic agent to an antibody that immunospecificallybinds to a 158P3D2 epitope, and, exposing the cell to the antibody-agentconjugate. Another illustrative embodiment is a method of treating anindividual suspected of suffering from metastasized cancer, comprising astep of administering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

[0299] Cancer immunotherapy using anti-158P3D2 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11: 117-127). Some therapeutic approaches involve conjugationof naked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 158P3D2 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

[0300] Although 158P3D2 antibody therapy is useful for all stages ofcancer, antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

[0301] Although 158P3D2 antibody therapy is useful for all stages ofcancer, antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

[0302] Cancer patients can be evaluated for the presence and level of158P3D2 expression, preferably using immunohistochemical assessments oftumor tissue, quantitative 158P3D2 imaging, or other techniques thatreliably indicate the presence and degree of 158P3D2 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

[0303] Anti-158P3D2 monoclonal antibodies that treat prostate and othercancers include those that initiate a potent immune response against thetumor or those that are directly cytotoxic. In this regard, anti-158P3D2monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-158P3D2 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 158P3D2. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-158P3D2 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

[0304] In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 158P3D2antigen with high affinity but exhibit low or no antigenicity in thepatient.

[0305] Therapeutic methods of the invention contemplate theadministration of single anti-158P3D2 mAbs as well as combinations, orcocktails, of different mAbs. Such mAb cocktails can have certainadvantages inasmuch as they contain mAbs that target different epitopes,exploit different effector mechanisms or combine directly cytotoxic mAbswith mAbs that rely on immune effector functionality. Such mAbs incombination can exhibit synergistic therapeutic effects. In addition,anti-158P3D2 mAbs can be administered concomitantly with othertherapeutic modalities, including but not limited to variouschemotherapeutic agents, androgen-blockers, immune modulators (e.g.,IL-2, GM-CSF), surgery or radiation. The anti-158P3D2 mAbs areadministered in their “naked” or unconjugated form, or can have atherapeutic agent(s) conjugated to them.

[0306] Anti-158P3D2 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-158P3D2antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3,4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

[0307] Based on clinical experience with the Herceptin™ mAb in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents anacceptable dosing regimen. Preferably, the initial loading dose isadministered as a 90 minute or longer infusion. The periodic maintenancedose is administered as a 30 minute or longer infusion, provided theinitial dose was well tolerated. As appreciated by those of skill in theart, various factors can influence the ideal dose regimen in aparticular case. Such factors include, for example, the binding affinityand half life of the Ab or mAbs used, the degree of 158P3D2 expressionin the patient, the extent of circulating shed 158P3D2 antigen, thedesired steady-state antibody concentration level, frequency oftreatment, and the influence of chemotherapeutic or other agents used incombination with the treatment method of the invention, as well as thehealth status of a particular patient.

[0308] Optionally, patients should be evaluated for the levels of158P3D2 in a given sample (e.g. the levels of circulating 158P3D2antigen and/or 158P3D2 expressing cells) in order to assist in thedetermination of the most effective dosing regimen, etc. Suchevaluations are also used for monitoring purposes throughout therapy,and are useful to gauge therapeutic success in combination with theevaluation of other parameters (for example, urine cytology and/orImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSAlevels in prostate cancer therapy).

[0309] Anti-idiotypic anti-158P3D2 antibodies can also be used inanti-cancer therapy as a vaccine for inducing an immune response tocells expressing a 158P3D2-related protein. In particular, thegeneration of anti-idiotypic antibodies is well known in the art; thismethodology can readily be adapted to generate anti-idiotypicanti-158P3D2 antibodies that mimic an epitope on a 158P3D2-relatedprotein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40;Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996,Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibodycan be used in cancer vaccine strategies.

[0310] X.C.) 158P3D2 as a Target for Cellular Immune Responses

[0311] Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

[0312] Carriers that can be used with vaccines of the invention are wellknown in the art, and include, e.g., thyroglobulin, albumins such ashuman serum albumin, tetanus toxoid, polyamino acids such as polyL-lysine, poly L-glutamic acid, influenza, hepatitis B virus coreprotein, and the like. The vaccines can contain a physiologicallytolerable (i.e., acceptable) diluent such as water, or saline,preferably phosphate buffered saline. The vaccines also typicallyinclude an adjuvant. Adjuvants such as incomplete Freund's adjuvant,aluminum phosphate, aluminum hydroxide, or alum are examples ofmaterials well known in the art. Additionally, as disclosed herein, CTLresponses can be primed by conjugating peptides of the invention tolipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS).Moreover, an adjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis J. Immunol. 165:539-547 (2000))

[0313] Upon immunization with a peptide composition in accordance withthe invention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 158P3D2 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

[0314] In some embodiments, it may be desirable to combine the class Ipeptide components with components that induce or facilitateneutralizing antibody and or helper T cell responses directed to thetarget antigen. A preferred embodiment of such a composition comprisesclass I and class II epitopes in accordance with the invention. Analternative embodiment of such a composition comprises a class I and/orclass II epitope in accordance with the invention, along with a crossreactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.)molecule (described e.g., in U.S. Pat. No. 5,736,142).

[0315] A vaccine of the invention can also include antigen-presentingcells (APC), such as dendritic cells (DC), as a vehicle to presentpeptides of the invention. Vaccine compositions can be created in vitro,following dendritic cell mobilization and harvesting, whereby loading ofdendritic cells occurs in vitro. For example, dendritic cells aretransfected, e.g., with a minigene in accordance with the invention, orare pulsed with peptides. The dendritic cell can then be administered toa patient to elicit immune responses in vivo. Vaccine compositions,either DNA- or peptide-based, can also be administered in vivo incombination with dendritic cell mobilization whereby loading ofdendritic cells occurs in vivo.

[0316] Preferably, the following principles are utilized when selectingan array of epitopes for inclusion in a polyepitopic composition for usein a vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

[0317] 1.) Epitopes are selected which, upon administration, mimicimmune responses that have been observed to be correlated with tumorclearance. For HLA Class I this includes 3-4 epitopes that come from atleast one tumor associated antigen (TAA). For HLA Class II a similarrationale is employed; again 3-4 epitopes are selected from at least oneTAA (see, e.g. Rosenberg et al., Science 278:1447-1450). Epitopes fromone TAA may be used in combination with epitopes from one or moreadditional TAAs to produce a vaccine that targets tumors with varyingexpression patterns of frequently-expressed TAAs.

[0318] 2.) Epitopes are selected that have the requisite bindingaffinity established to be correlated with immunogenicity: for HLA ClassI an IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II anIC₅₀ of 1000 nM or less.

[0319] 3.) Sufficient supermotif bearing-peptides, or a sufficient arrayof allele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

[0320] 4.) When selecting epitopes from cancer-related antigens it isoften useful to select analogs because the patient may have developedtolerance to the native epitope.

[0321] 5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

[0322] 6.) If a polyepitopic protein is created, or when creating aminigene, an objective is to generate the smallest peptide thatencompasses the epitopes of interest. This principle is similar, if notthe same as that employed when selecting a peptide comprising nestedepitopes. However, with an artificial polyepitopic peptide, the sizeminimization objective is balanced against the need to integrate anyspacer sequences between epitopes in the polyepitopic protein. Spaceramino acid residues can, for example, be introduced to avoid junctionalepitopes (an epitope recognized by the immune system, not present in thetarget antigen, and only created by the man-made juxtaposition ofepitopes), or to facilitate cleavage between epitopes and therebyenhance epitope presentation. Junctional epitopes are generally to beavoided because the recipient may generate an immune response to thatnon-native epitope. Of particular concern is a junctional epitope thatis a “dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

[0323] 7.) Where the sequences of multiple variants of the same targetprotein are present, potential peptide epitopes can also be selected onthe basis of their conservancy. For example, a criterion for conservancymay define that the entire sequence of an HLA class I binding peptide orthe entire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

[0324] X.C.1.) Minigene Vaccines

[0325] A number of different approaches are available which allowsimultaneous delivery of multiple epitopes. Nucleic acids encoding thepeptides of the invention are a particularly useful embodiment of theinvention. Epitopes for inclusion in a minigene are preferably selectedaccording to the guidelines set forth in the previous section. Apreferred means of administering nucleic acids encoding the peptides ofthe invention uses minigene constructs encoding a peptide comprising oneor multiple epitopes of the invention.

[0326] The use of multi-epitope minigenes is described below and in,Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J.L., J. Virol 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822,1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al.,Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 158P3D2, the PADRE(Duniversal helper T cell epitope (or multiple HTL epitopes from 158P3D2),and an endoplasmic reticulum-translocating signal sequence can beengineered. A vaccine may also comprise epitopes that are derived fromother TAAs.

[0327] The immunogenicity of a multi-epitopic minigene can be confirmedin transgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

[0328] For example, to create a DNA sequence encoding the selectedepitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes may be reverse translated. A human codon usagetable can be used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

[0329] The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

[0330] Standard regulatory sequences well known to those of skill in theart are preferably included in the vector to ensure expression in thetarget cells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0331] Additional vector modifications may be desired to optimizeminigene expression and immunogenicity. In some cases, introns arerequired for efficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

[0332] Once an expression vector is selected, the minigene is clonedinto the polylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

[0333] In addition, immunostimulatory sequences (ISSs or CpGs) appear toplay a role in the immunogenicity of DNA vaccines. These sequences maybe included in the vector, outside the minigene coding sequence, ifdesired to enhance immunogenicity.

[0334] In some embodiments, a bi-cistronic expression vector whichallows production of both the minigene-encoded epitopes and a secondprotein (included to enhance or decrease immunogenicity) can be used.Examples of proteins or polypeptides that could beneficially enhance theimmune response if co-expressed include cytokines (e.g., IL-2, IL-12,GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatorymolecules, or for HTL responses, pan-DR binding proteins (PADRE™,Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined tointracellular targeting signals and expressed separately from expressedCTL epitopes; this allows direction of the HTL epitopes to a cellcompartment different than that of the CTL epitopes. If required, thiscould facilitate more efficient entry of HTL epitopes into the HLA classII pathway, thereby improving HTL induction. In contrast to HTL or CTLinduction, specifically decreasing the immune response by co-expressionof immunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

[0335] Therapeutic quantities of plasmid DNA can be produced forexample, by fermentation in E. coli, followed by purification. Aliquotsfrom the working cell bank are used to inoculate growth medium, andgrown to saturation in shaker flasks or a bioreactor according towell-known techniques. Plasmid DNA can be purified using standardbioseparation technologies such as solid phase anion-exchange resinssupplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiledDNA can be isolated from the open circular and linear forms using gelelectrophoresis or other methods.

[0336] Purified plasmid DNA can be prepared for injection using avariety of formulations. The simplest of these is reconstitution oflyophilized DNA in sterile phosphate-buffer saline (PBS). This approach,known as “naked DNA,” is currently being used for intramuscular (IM)administration in clinical trials. To maximize the immunotherapeuticeffects of minigene DNA vaccines, an alternative method for formulatingpurified plasmid DNA may be desirable. A variety of methods have beendescribed, and new techniques may become available. Cationic lipids,glycolipids, and fusogenic liposomes can also be used in the formulation(see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite,BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309;and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). Inaddition, peptides and compounds referred to collectively as protective,interactive, non-condensing compounds (PINC) could also be complexed topurified plasmid DNA to influence variables such as stability,intramuscular dispersion, or trafficking to specific organs or celltypes.

[0337] Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

[0338] In vivo immunogenicity is a second approach for functionaltesting of minigene DNA formulations. Transgenic mice expressingappropriate human HLA proteins are immunized with the DNA product. Thedose and route of administration are formulation dependent (e.g., IM forDNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-onedays after immunization, splenocytes are harvested and restimulated forone week in the presence of peptides encoding each epitope being tested.Thereafter, for CTL effector cells, assays are conducted for cytolysisof peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

[0339] Alternatively, the nucleic acids can be administered usingballistic delivery as described, for instance, in U.S. Pat. No.5,204,253. Using this technique, particles comprised solely of DNA areadministered. In a further alternative embodiment, DNA can be adhered toparticles, such as gold particles.

[0340] Minigenes can also be delivered using other bacterial or viraldelivery systems well known in the art, e.g., an expression constructencoding epitopes of the invention can be incorporated into a viralvector such as vaccinia.

[0341] X.C.2.) Combinations of CTL Peptides with Helper Peptides

[0342] Vaccine compositions comprising CTL peptides of the invention canbe modified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

[0343] For instance, the ability of a peptide to induce CTL activity canbe enhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

[0344] In certain embodiments, the T helper peptide is one that isrecognized by T helper cells present in a majority of a geneticallydiverse population. This can be accomplished by selecting peptides thatbind to many, most, or all of the HLA class II molecules. Examples ofsuch amino acid bind many HLA Class II molecules include sequences fromantigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE;SEQ ID NO: ______, Plasmodium falciparum circumsporozoite (CS) proteinat positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: ______), andStreptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQID NO: ______. Other examples include peptides bearing a DR 1-4-7supermotif, or either of the DR3 motifs.

[0345] Alternatively, it is possible to prepare synthetic peptidescapable of stimulating T helper lymphocytes, in a loosely HLA-restrictedfashion, using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: ______, where “X” iseither cyclohexylalanine, phenylalanine, or tyrosine, and a is eitherD-alanine or L-alanine, has been found to bind to most HLA-DR alleles,and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type. An alternative of a pan-DRbinding epitope comprises all “L” natural amino acids and can beprovided in the form of nucleic acids that encode the epitope.

[0346] HTL peptide epitopes can also be modified to alter theirbiological properties. For example, they can be modified to includeD-amino acids to increase their resistance to proteases and thus extendtheir serum half life, or they can be conjugated to other molecules suchas lipids, proteins, carbohydrates, and the like to increase theirbiological activity. For example, a T helper peptide can be conjugatedto one or more palmitic acid chains at either the amino or carboxyltermini.

[0347] X.C.3.) Combinations of CTL Peptides with T Cell Priming Agents

[0348] In some embodiments it may be desirable to include in thepharmaceutical compositions of the invention at least one componentwhich primes B lymphocytes or T lymphocytes. Lipids have been identifiedas agents capable of priming CTL in vivo. For example, palmitic acidresidues can be attached to the ε- and α-amino groups of a lysineresidue and then linked, e.g. via one or more linking residues such asGly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be administered either directly in a micelleor particle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0349] As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

[0350] X.C.4.) Vaccine Compositions Comprising DC Pulsed with CTL and/orHTL Peptides

[0351] An embodiment of a vaccine composition in accordance with theinvention comprises ex vivo administration of a cocktail ofepitope-bearing peptides to PBMC, or isolated DC therefrom, from thepatient's blood. A pharmaceutical to facilitate harvesting of DC can beused, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) orGM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusioninto patients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

[0352] The DC can be pulsed ex vivo with a cocktail of peptides, some ofwhich stimulate CTL responses to 158P3D2. Optionally, a helper T cell(HTL) peptide, such as a natural or artificial loosely restricted HLAClass II peptide, can be included to facilitate the CTL response. Thus,a vaccine in accordance with the invention is used to treat a cancerwhich expresses or overexpresses 158P3D2.

[0353] X.D.) Adoptive Immunotherapy

[0354] Antigenic 158P3D2-related peptides are used to elicit a CTLand/or HTL response ex vivo, as well. The resulting CTL or HTL cells,can be used to treat tumors in patients that do not respond to otherconventional forms of therapy, or will not respond to a therapeuticvaccine peptide or nucleic acid in accordance with the invention. Exvivo CTL or HTL responses to a particular antigen are induced byincubating in tissue culture the patient's, or genetically compatible,CTL or HTL precursor cells together with a source of antigen-presentingcells (APC), such as dendritic cells, and the appropriate immunogenicpeptide. After an appropriate incubation time (typically about 7-28days), in which the precursor cells are activated and expanded intoeffector cells, the cells are infused back into the patient, where theywill destroy (CTL) or facilitate destruction (HTL) of their specifictarget cell (e.g., a tumor cell). Transfected dendritic cells may alsobe used as antigen presenting cells.

[0355] X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

[0356] Pharmaceutical and vaccine compositions of the invention aretypically used to treat and/or prevent a cancer that expresses oroverexpresses 158P3D2. In therapeutic applications, peptide and/ornucleic acid compositions are administered to a patient in an amountsufficient to elicit an effective B cell, CTL and/or HTL response to theantigen and to cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

[0357] For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 158P3D2. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

[0358] For therapeutic use, administration should generally begin at thefirst diagnosis of 158P3D2-associated cancer. This is followed byboosting doses until at least symptoms are substantially abated and fora period thereafter. The embodiment of the vaccine composition (i.e.,including, but not limited to embodiments such as peptide cocktails,polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulseddendritic cells) delivered to the patient may vary according to thestage of the disease or the patient's health status. For example, in apatient with a tumor that expresses 158P3D2, a vaccine comprising158P3D2-specific CTL may be more efficacious in killing tumor cells inpatient with advanced disease than alternative embodiments.

[0359] It is generally important to provide an amount of the peptideepitope delivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

[0360] The dosage for an initial therapeutic immunization generallyoccurs in a unit dosage range where the lower value is about 1, 5, 50,500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000;or 50,000 μg. Dosage values for a human typically range from about 500μg to about 50,000 μg per 70 kilogram patient. Boosting dosages ofbetween about 1.0 μg to about 50,000 μg of peptide pursuant to aboosting regimen over weeks to months may be administered depending uponthe patient's response and condition as determined by measuring thespecific activity of CTL and HTL obtained from the patient's blood.Administration should continue until at least clinical symptoms orlaboratory tests indicate that the neoplasia, has been eliminated orreduced and for a period thereafter. The dosages, routes ofadministration, and dose schedules are adjusted in accordance withmethodologies known in the art.

[0361] In certain embodiments, the peptides and compositions of thepresent invention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

[0362] The vaccine compositions of the invention can also be used purelyas prophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

[0363] The pharmaceutical compositions for therapeutic treatment areintended for parenteral, topical, oral, nasal, intrathecal, or local(e.g. as a cream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

[0364] A variety of aqueous carriers may be used, e.g., water, bufferedwater, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

[0365] The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

[0366] The concentration of peptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

[0367] A human unit dose form of a composition is typically included ina pharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

[0368] For antibodies, a treatment generally involves repeatedadministration of the anti-158P3D2 antibody preparation, via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. In general, doses in the range of 10-500 mg mAb per week areeffective and well tolerated. Moreover, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents anacceptable dosing regimen. As appreciated by those of skill in the art,various factors can influence the ideal dose in a particular case. Suchfactors include, for example, half life of a composition, the bindingaffinity of an Ab, the immunogenicity of a substance, the degree of158P3D2 expression in the patient, the extent of circulating shed158P3D2 antigen, the desired steady-state concentration level, frequencyof treatment, and the influence of chemotherapeutic or other agents usedin combination with the treatment method of the invention, as well asthe health status of a particular patient. Non-limiting preferred humanunit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg,700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certainembodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., withfollow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9,10 mg/kg body weight followed, e.g., in two, three or four weeks byweekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three orfour weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m²of body area weekly; 1-600 mg m² of body area weekly; 225-400 mg m² ofbody area weekly; these does can be followed by weekly doses for 2, 3,4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

[0369] In one embodiment, human unit dose forms of polynucleotidescomprise a suitable dosage range or effective amount that provides anytherapeutic effect. As appreciated by one of ordinary skill in the art atherapeutic effect depends on a number of factors, including thesequence of the polynucleotide, molecular weight of the polynucleotideand route of administration. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. Generally, for a polynucleotide of about 20bases, a dosage range may be selected from, for example, anindependently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kgup to an independently selected upper limit, greater than the lowerlimit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, adose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to10,000 mg/kg. Generally, parenteral routes of administration may requirehigher doses of polynucleotide compared to more direct application tothe nucleotide to diseased tissue, as do polynucleotides of increasinglength.

[0370] In one embodiment, human unit dose forms of T-cells comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art, atherapeutic effect depends on a number of factors. Dosages are generallyselected by the physician or other health care professional inaccordance with a variety of parameters known in the art, such asseverity of symptoms, history of the patient and the like. A dose may beabout 10⁴ cells to about 10⁶ cells, about 10⁶ cells to about 10⁸ cells,about 10⁸ to about 10¹¹ cells, or about 10⁸ to about 5×10¹⁰ cells. Adose may also about 10⁶ cells/m² to about 10¹⁰ cells/m², or about 10⁶cells/m² to about 10⁸ cells/m².

[0371] Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0372] For targeting cells of the immune system, a ligand to beincorporated into the liposome can include, e.g., antibodies orfragments thereof specific for cell surface determinants of the desiredimmune system cells. A liposome suspension containing a peptide may beadministered intravenously, locally, topically, etc. in a dose whichvaries according to, inter alia, the manner of administration, thepeptide being delivered, and the stage of the disease being treated.

[0373] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

[0374] For aerosol administration, immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are about 0.01%-20% by weight,preferably about 1%-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from about 6to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural -glycerides may be employed. The surfactant mayconstitute about 0.1%-20% by weight of the composition, preferably about0.25-5%. The balance of the composition is ordinarily propellant. Acarrier can also be included, as desired, as with, e.g., lecithin forintranasal delivery.

[0375] XI.) Diagnostic and Prognostic Embodiments of 158P3D2.

[0376] As disclosed herein, 158P3D2 polynucleotides, polypeptides,reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

[0377] 158P3D2 can be analogized to a prostate associated antigen PSA,the archetypal marker that has been used by medical practitioners foryears to identify and monitor the presence of prostate cancer (see,e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al.,J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med July 1999 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of158P3D2 polynucleotides and polypeptides (as well as 158P3D2polynucleotide probes and anti-158P3D2 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

[0378] Typical embodiments of diagnostic methods which utilize the158P3D2 polynucleotides, polypeptides, reactive T cells and antibodiesare analogous to those methods from well-established diagnostic assayswhich employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 158P3D2 polynucleotides described herein can be utilized in the sameway to detect 158P3D2 overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the158P3D2 polypeptides described herein can be utilized to generateantibodies for use in detecting 158P3D2 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

[0379] Specifically, because metastases involves the movement of cancercells from an organ of origin (such as the lung or prostate gland etc.)to a different area of the body (such as a lymph node), assays whichexamine a biological sample for the presence of cells expressing 158P3D2polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 158P3D2-expressing cells (lymph node) is found tocontain 158P3D2-expressing cells such as the 158P3D2 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

[0380] Alternatively 158P3D2 polynucleotides and/or polypeptides can beused to provide evidence of cancer, for example, when cells in abiological sample that do not normally express 158P3D2 or express158P3D2 at a different level are found to express 158P3D2 or have anincreased expression of 158P3D2 (see, e.g., the 158P3D2 expression inthe cancers listed in Table I and in patient samples etc. shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 158P3D2) such as PSA, PSCA etc. (see, e.g., Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

[0381] Just as PSA polynucleotide fragments and polynucleotide variantsare employed by skilled artisans for use in methods of monitoring PSA,158P3D2 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 158P3D2 polynucleotidefragment is used as a probe to show the expression of 158P3D2 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther.November-December 1996 11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubel et al eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g., a158P3D2 polynucleotide shown in FIG. 2 or variant thereof) underconditions of high stringency.

[0382] Furthermore, PSA polypeptides which contain an epitope that canbe recognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 158P3D2 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 158P3D2biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 158P3D2 polypeptide shown in FIG. 3).

[0383] As shown herein, the 158P3D2 polynucleotides and polypeptides (aswell as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies orT cells used to identify the presence of these molecules) exhibitspecific properties that make them useful in diagnosing cancers such asthose listed in Table I. Diagnostic assays that measure the presence of158P3D2 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 158P3D2polynucleotides and polypeptides (as well as the 158P3D2 polynucleotideprobes and anti-158P3D2 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

[0384] Finally, in addition to their use in diagnostic assays, the158P3D2 polynucleotides disclosed herein have a number of otherutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in the chromosomal region to whichthe 158P3D2 gene maps (see Example 3 below). Moreover, in addition totheir use in diagnostic assays, the 158P3D2-related proteins andpolynucleotides disclosed herein have other utilities such as their usein thc forensic analysis of tissues of unknown origin (see, e.g.,Takahama K Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).

[0385] Additionally, 158P3D2-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 158P3D2. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 158P3D2 antigen. Antibodies or othermolecules that react with 158P3D2 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

[0386] XII.) Inhibition of 158P3D2 Protein Function

[0387] The invention includes various methods and compositions forinhibiting the binding of 158P3D2 to its binding partner or itsassociation with other protein(s) as well as methods for inhibiting158P3D2 function.

[0388] XII.A.) Inhibition of 158P3D2 with Intracellular Antibodies

[0389] In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 158P3D2 are introduced into 158P3D2expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-158P3D2 antibody is expressed intracellularly,binds to 158P3D2 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

[0390] Single chain antibodies comprise the variable domains of theheavy and light chain joined by a flexible linker polypeptide, and areexpressed as a single polypeptide. Optionally, single chain antibodiesare expressed as a single chain variable region fragment joined to thelight chain constant region. Well-known intracellular traffickingsignals are engineered into recombinant polynucleotide vectors encodingsuch single chain antibodies in order to precisely target the intrabodyto the desired intracellular compartment. For example, intrabodiestargeted to the endoplasmic reticulum (ER) are engineered to incorporatea leader peptide and, optionally, a C-terminal ER retention signal, suchas the KDEL amino acid motif Intrabodies intended to exert activity inthe nucleus are engineered to include a nuclear localization signal.Lipid moieties are joined to intrabodies in order to tether theintrabody to the cytosolic side of the plasma membrane. Intrabodies canalso be targeted to exert function in the cytosol. For example,cytosolic intrabodies are used to sequester factors within the cytosol,thereby preventing them from being transported to their natural cellulardestination.

[0391] In one embodiment, intrabodies are used to capture 158P3D2 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals arc engineered into such 158P3D2 intrabodies in orderto achieve the desired targeting. Such 158P3D2 intrabodies are designedto bind specifically to a particular 158P3D2 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 158P3D2protein are used to prevent 158P3D2 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 158P3D2 from forming transcription complexeswith other factors).

[0392] In order to specifically direct the expression of suchintrabodies to particular cells, the transcription of the intrabody isplaced under the regulatory control of an appropriate tumor-specificpromoter and/or enhancer. In order to target intrabody expressionspecifically to prostate, for example, the PSA promoter and/orpromoter/enhancer can be utilized (See, for example, U.S. Pat. No.5,919,652 issued Jul. 6, 1999).

[0393] XII.B.) Inhibition of 158P3D2 with Recombinant Proteins

[0394] In another approach, recombinant molecules bind to 158P3D2 andthereby inhibit 158P3D2 function. For example, these recombinantmolecules prevent or inhibit 158P3D2 from accessing/binding to itsbinding partner(s) or associating with other protein(s). Suchrecombinant molecules can, for example, contain the reactive part(s) ofa 158P3D2 specific antibody molecule. In a particular embodiment, the158P3D2 binding domain of a 158P3D2 binding partner is engineered into adimeric fusion protein, whereby the fusion protein comprises two 158P3D2ligand binding domains linked to the Fc portion of a human IgG, such ashuman IgG1. Such IgG portion can contain, for example, the CH2 and CH3domains and the hinge region, but not the CH1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 158P3D2, whereby thedimeric fusion protein specifically binds to 158P3D2 and blocks 158P3D2interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

[0395] XH.C.) Inhibition of 158P3D2 Transcription or Translation

[0396] The present invention also comprises various methods andcompositions for inhibiting the transcription of the 158P3D2 gene.Similarly, the invention also provides methods and compositions forinhibiting the translation of 158P3D2 mRNA into protein.

[0397] In one approach, a method of inhibiting the transcription of the158P3D2 gene comprises contacting the 158P3D2 gene with a 158P3D2antisense polynucleotide. In another approach, a method of inhibiting158P3D2 mRNA translation comprises contacting a 158P3D2 mRNA with anantisense polynucleotide. In another approach, a 158P3D2 specificribozyme is used to cleave a 158P3D2 message, thereby inhibitingtranslation. Such antisense and ribozyme based methods can also bedirected to the regulatory regions of the 158P3D2 gene, such as 158P3D2promoter and/or enhancer elements. Similarly, proteins capable ofinhibiting a 158P3D2 gene transcription factor are used to inhibit158P3D2 mRNA transcription. The various polynucleotides and compositionsuseful in the aforementioned methods have been described above. The useof antisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

[0398] Other factors that inhibit the transcription of 158P3D2 byinterfering with 158P3D2 transcriptional activation are also useful totreat cancers expressing 158P3D2. Similarly, factors that interfere with158P3D2 processing are useful to treat cancers that express 158P3D2.Cancer treatment methods utilizing such factors are also within thescope of the invention.

[0399] XII.D.) General Considerations for Therapeutic Strategies

[0400] Gene transfer and gene therapy technologies can be used todeliver therapeutic polynucleotide molecules to tumor cells synthesizing158P3D2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodiesand other 158P3D2 inhibitory molecules). A number of gene therapyapproaches are known in the art. Recombinant vectors encoding 158P3D2antisense polynucleotides, ribozymes, factors capable of interferingwith 158P3D2 transcription, and so forth, can be delivered to targettumor cells using such gene therapy approaches.

[0401] The above therapeutic approaches can be combined with any one ofa wide variety of surgical, chemotherapy or radiation therapy regimens.The therapeutic approaches of the invention can enable the use ofreduced dosages of chemotherapy (or other therapies) and/or lessfrequent administration, an advantage for all patients and particularlyfor those that do not tolerate the toxicity of the chemotherapeuticagent well.

[0402] The anti-tumor activity of a particular composition (e.g.,antisense, ribozyme, intrabody), or a combination of such compositions,can be evaluated using various in vitro and in vivo assay systems. Invii-o assays that evaluate therapeutic activity include cell growthassays, soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 158P3D2 to a bindingpartner, etc.

[0403] In vivo, the effect of a 158P3D2 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

[0404] In vivo assays that evaluate the promotion of apoptosis areuseful in evaluating therapeutic compositions. In one embodiment,xenografts from tumor bearing mice treated with the therapeuticcomposition can be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

[0405] The therapeutic compositions used in the practice of theforegoing methods can be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material that when combined with the therapeuticcomposition retains the anti-tumor function of the therapeuticcomposition and is generally non-reactive with the patient's immunesystem. Examples include, but are not limited to, any of a number ofstandard pharmaceutical carriers such as sterile phosphate bufferedsaline solutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

[0406] Therapeutic formulations can be solubilized and administered viaany route capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

[0407] Dosages and administration protocols for the treatment of cancersusing the foregoing methods will vary with the method and the targetcancer, and will generally depend on a number of other factorsappreciated in the art.

[0408] XIII.) Kits

[0409] For use in the diagnostic and therapeutic applications describedherein, kits are also within the scope of the invention. Such kits cancomprise a carrier, package or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin the method. For example, the container(s) can comprise a probe thatis or can be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 158P3D2-related protein or a 158P3D2 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, fluorescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

[0410] The kit of the invention will typically comprise the containerdescribed above and one or more other containers comprising materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

[0411] A label can be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and can also indicate directions for either in vivo or invitro use, such as those described above. Directions and or otherinformation can also be included on an insert which is included with thekit.

EXAMPLES

[0412] Various aspects of the invention are further described andillustrated by way of the several examples that follow, none of whichare intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene

[0413] To isolate genes that are over-expressed in bladder cancer,Suppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom bladder cancer tissues, including invasive transitional cellcarcinoma. The 158P3D2 SSH cDNA sequence was derived from a bladdercancer pool minus normal bladder cDNA subtraction. Included in thedriver were also cDNAs derived from 9 other normal tissues. The 158P3D2cDNA was identified as highly expressed in the bladder cancer tissuepool, with lower expression seen in a restricted set of normal tissues.

[0414] The SSH DNA sequence of 312 bp (FIG. 1) shows identity to thefer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2 cDNA clone158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealingan ORF of 328 amino acids (FIG. 2 and FIG. 3).

[0415] Amino acid sequence analysis of 158P3D2 reveals 100% identityover 328 amino acid region to dJ47704. 1. 1, a novel protein similar tootoferlin and dysferlin, isoform 1 protein (GenBank AccessionCAB89410.1|, FIG. 4B).

[0416] The 158P3D2 protein has a transmembrane domain of 23 residuesbetween amino acids 292-313 predicted by the SOSUI Signal program(http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosuisignal_submit.html).

[0417] Materials and Methods

[0418] Human Tissues:

[0419] The patient cancer and normal tissues were purchased fromdifferent sources such as the NDRI (Philadelphia, Pa.). mRNA for somenormal tissues were purchased from Clontech, Palo Alto, Calif.

[0420] RNA Isolation:

[0421] Tissues were homogenized in Trizol reagent (Life Technologies,Gibco BRL) using 10 ml/ g tissue isolate total RNA. Poly A RNA waspurified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits.Total and mRNA were quantified by spectrophotometric analysis (O.D.260/280 nm) and analyzed by gel electrophoresis.

[0422] Oligonucleotides:

[0423] The following HPLC purified oligonucleotides were used.

[0424] DPNCDN (cDNA Synthesis Primer):

[0425] 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: ______) DPNCDN (cDNA synthesisprimer): (SEQ ID NO:_) 5′TTTTGATCAAGTT₃₀3′ Adaptor 1: (SEQ ID NO:_)5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO:_)3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO:_)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO:_)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO:_) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO:_) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO:_) 5′AGCGTGGTCGCGGCCGAGGA3′

[0426] PCR Primer 1:

[0427] 5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: ______)

[0428] Nested Primer (NP)1:

[0429] 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: ______)

[0430] Nested Primer (NP)₂:

[0431] 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: ______)

[0432] Suppression Subtractive Hybridization:

[0433] Suppression Subtractive Hybridization (SSH) was used to identifycDNAs corresponding to genes that may be differentially expressed inbladder cancer. The SSH reaction utilized cDNA from bladder cancer andnormal tissues.

[0434] The gene 15 8P3D2 sequence was derived from a bladder cancer poolminus normal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) wasidentified.

[0435] The cDNA derived from of pool of normal bladder tissues was usedas the source of the “driver” cDNA, while the cDNA from a pool ofbladder cancer tissues was used as the source of the “tester” cDNA.Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)+ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

[0436] Driver cDNA was generated by combining in a 1:1 ratio Dpn IIdigested cDNA from the relevant tissue source (see above) with a mix ofdigested cDNAs derived from the nine normal tissues: stomach, skeletalmuscle, lung, brain, liver, kidney, pancreas, small intestine, andheart.

[0437] Tester cDNA was generated by diluting 1 μl of Dpn II digestedcDNA from the relevant tissue source (see above) (400 ng) in 5 μl ofwater. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl ofAdaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in atotal volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase(CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heatingat 72° C. for 5 min.

[0438] The first hybridization was performed by adding 1.5 μl (600 ng)of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

[0439] PCR Amplification, Cloning and Sequencing of Gene FragmentsGenerated from SSH:

[0440] To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer I (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10×reaction buffer (CLONTECH) and0.5 μl 50×Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR I was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 see, 66° C.for 30 see, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

[0441] The PCR products were inserted into pCR2.1 using the T/A vectorcloning kit (Invitrogen). Transformed E. coli were subjected toblue/white and ampicillin selection. White colonies were picked andarrayed into 96 well plates and were grown in liquid culture overnight.To identify inserts, PCR amplification was performed on 1 ml ofbacterial culture using the conditions of PCR1 and NP1 and NP2 asprimers. PCR products were analyzed using 2% agarose gelelectrophoresis.

[0442] Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbest, and NCI-CGAP databases.

[0443] RT-PCR Expression Analysis:

[0444] First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

[0445] Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5 ′atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO: ______) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: ______) toamplify β-actin. First strand cDNA (5 μl) were amplified in a totalvolume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCRbuffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3) and1×-Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can beremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: Initial denaturation can be at 94° C.for 15 see, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C.for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried outfor 2 min. After agarose gel electrophoresis, the band intensities ofthe 283 b.p. β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization can be required to achieveequal band intensities in all tissues after 22 cycles of PCR.

[0446] To determine expression levels of the 158P3D2 gene, 5 μl ofnormalized first strand cDNA were analyzed by PCR using 26, and 30cycles of amplification. Semi-quantitative expression analysis can beachieved by comparing the PCR products at cycle numbers that give lightband intensities. The primers used for RT-PCR were designed using the158P3D2 SSH sequence and are listed below:

[0447] 158P3D2.1

[0448] 5′CATCTATGTGAAGAGCTGGGTGAA 3′ (SEQ ID NO: )

[0449] 158P3D2.2

[0450] 5′ AGGTAGTCAAAGCGGAACACAAAG 3′ (SEQ ID NO: )

[0451] A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Results show strong expression of158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasispool. Expression of 158P3D2 is also detected in colon cancer pool, lungcancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pooland prostate metastases to lymph node, and vital pool 2, but not vitalpool 1.

Example 2 Full Length Cloning of 158P3D2

[0452] The 158P3D2 SSH cDNA sequence was derived from a bladder cancerpool minus normal bladder cDNA subtraction. The SSH cDNA sequence(FIG. 1) was designated 158P3D2. The full-length cDNA clone 158P3D2 v.1clone 158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were cloned from bladdercancer pool cDNA.

[0453] The SSH DNA sequence of 312 bp (FIG. 1) shows identity to thefer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2 cDNA clone158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealingan ORF of 328 amino acids (FIG. 2 and FIG. 3).

[0454] Amino acid sequence analysis of 158P3D2 reveals 100% identityover 328 amino acid region to dJ47704.1.1, a novel protein similar tootoferlin and dysferlin, isoform 1 protein (GenBank AccessionCAB89410.1|, FIG. 4B).

[0455] The 158P3D2 protein has a transmembrane domain of 23 residuesbetween amino acids 292-313 predicted by the SOSUI Signal program(http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosuisignal_submit.html).

Example 3 Chromosomal Mapping of 158P3D2

[0456] Chromosomal localization can implicate genes in diseasepathogenesis. Several chromosome mapping approaches are availableincluding fluorescent in situ hybridization (FISH), human/hamsterradiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22;Research Genetics, Huntsville Al), human-rodent somatic cell hybridpanels such as is available from the Coriell Institute (Camden, N.J.),and genomic viewers utilizing BLAST homologies to sequenced and mappedgenomic clones (NCBI, Bethesda, Md.).

[0457] 158P3D2 maps to chromosome 8, using 158P3D2 sequence and the NCBIBLAST tool:(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4 Expression Analysis of 158P3D2 in Normal Tissues and PatientSpecimens

[0458] Expression analysis by RT-PCR demonstrated that 158P3D2 isstrongly expressed in bladder cancer patient specimens (FIG. 14). Firststrand cDNA was prepared from vital pool 1 (liver, lung and kidney),vital pool 2 (pancreas, colon and stomach), prostate cancer metastasisto lymph node from 2 different patients, prostate cancer pool, bladdercancer pool, kidney cancer pool, colon cancer pool, lung cancer pool,ovary cancer pool, breast cancer pool, cancer metastasis pool, andpancreas cancer pool. Normalization was performed by PCR using primersto actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, wasperformed at 26 and 30 cycles of amplification. Results show strongexpression of 158P3D2 in bladder cancer pool, kidney cancer pool andcancer metastasis pool. Expression of 158P3D2 is also detected in coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,pancreas cancer pool and prostate metastases to lymph node, and vitalpool 2, but not vital pool 1.

[0459] Northern blot analysis of 158P3D2 in 16 human normal tissues isshown in FIG. 15. An approximately 8 kb transcript is detectedexclusively in placenta. Extensive analysis of expression of 158P3D2 in76 human tissues shows restricted expression of 158P3D2 in placenta andstomach (FIG. 16).

[0460] Expression of 158P3D2 in patient cancer specimens and humannormal tissues is shown in FIG. 17. RNA was extracted from a pool ofthree bladder cancers, as well as from normal prostate (NP), normalbladder (NB), normal kidney (NK), normal colon (NC), normal lung (NL)and normal breast (NBr). Northern blot with 10 ug of total RNA/lane wasprobed with 158P3D2 sequence. The results show expression of 158P3D2 inthe bladder cancer pool but not in the normal tissues tested. Analysisof individual patient specimens shows strong expression of 158P3D2 in 8different bladder cancer tissues tested (FIG. 18). Presence of 158P3D2transcript is also detected in the bladder cancer cell line SCaBER. Theexpression observed in normal adjacent tissue (isolated from diseasedtissues) but not in normal tissue, isolated from healthy donors, mayindicate that these tissues are not fully normal and that 158P3D2 may beexpressed in early stage tumors.

[0461] The restricted expression of 158P3D2 in normal tissues and theexpression detected in bladder cancer, prostate cancer pool, kidneycancer pool, colon cancer pool, lung cancer pool, ovary cancer pool,breast cancer pool, pancreas cancer pool and cancer metastases suggestthat 158P3D2 is a potential therapeutic target and a diagnostic markerfor human cancers.

Example 5 Transcript Variants of 158P3D2

[0462] Transcript variants are variants of matured mRNA from the samegene by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or mayencode proteins with different functions, and may be expressed in thesame tissue at the same time, or at different tissue, or at differenttimes, proteins encoded by transcript variants can have similar ordifferent cellular or extracellular localizations, i.e., be secreted.

[0463] Transcript variants are identified by a variety of art-acceptedmethods. For example, alternative transcripts and splice variants areidentified through full-length cloning experiments, or by use offull-length transcript and EST sequences. First, all human ESTs weregrouped into clusters which show direct or indirect identity with eachother. Second, ESTs in the same cluster were further grouped intosub-clusters and assembled into a consensus sequence. The original genesequence is compared to the consensus sequence(s) or other full-lengthsequences. Each consensus sequence is a potential splice variant forthat gene (see, e.g.,http://www.doubletwist.com/products/c11_agentsOverview.jhtml). Even whena variant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

[0464] Moreover, computer programs are available in the art thatidentify transcript variants based on genomic sequences. Genomic-basedtranscript variant identification programs include FgenesH (A. Salamovand V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,”Genome Research. April 2000; 10(4):516-22); Grail(http://compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan(http://genes.mit.edu/GENSCAN.html). For a general discussion of splicevariant identification protocols see., e.g., Southan, C., A genomicperspective on human proteases, FEBS Lett. Jun. 8, 2001; 498(2-3):214-8;de Souza, S. J., et al., Identification of human chromosome 22transcribed sequences with ORF expressed sequence tags, Proc. Natl. AcadSci USA. Nov. 7, 2000; 97(23):12690-3.

[0465] To further confirm the parameters of a transcript variant, avariety of techniques are available in the art, such as full-lengthcloning, proteomic validation, PCR-based validation, and 5′ RACEvalidation, etc. (see e.g., Proteomic Validation: Brennan, S. O., etal., Albumin banks peninsula: a new termination variant characterized byelectrospray mass spectrometry, Biochem Biophys Acta. Aug. 17,1999;1433(1-2):321-6; Ferranti P, et al., Differential splicing ofpre-messenger RNA produces multiple forms of mature caprinealpha(s1)-casein, Eur J. Biochem. Oct. 1, 1997;249(1):1-7. For PCR-basedValidation: Wellmann S, et al., Specific reverse transcription-PCRquantification of vascular endothelial growth factor (VEGF) splicevariants by LightCycler technology, Clin Chem. April 2001; 47(4):654-60;Jia, H. P., et al., Discovery of new human beta-defensins using agenomics-based approach, Gene. Jan. 24, 2001; 263(1-2):211-8. ForPCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization ofthe murine reduced folate carrier gene and identification of variantsplice forms, Biochem Biophys Acta. Aug. 7, 1997; 1353(2): 191-8).

[0466] It is known in the art that genomic regions are modulated incancers. When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 158P3D2 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 158P3D2 may also be involved in cancer in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

[0467] The exon composition of the original transcript, designated as158P3D2 var1, is shown in FIG. 13 and Table XXIIIA. Using thefull-length gene and EST sequences, one alternative transcript wasidentified, designated as 158P3D2 var2, which is also shown in FIG. 13and Table XXIIIB. Transcript variant 158P3D2 var2 has two potential openreading frames and two protein products, designated as 158P3D2 var2a and158P3D2 var2b. FIG. 13 shows the schematic alignment of exons of the twotranscripts. Potentially, each different combination of exons in spatialorder, e.g. exons 1, 2, 3, 4 and 7, can be a splice variant.

[0468] Tables XXIV through XXVII are set forth herein on avariant-by-variant basis. Table XXIV shows nucleotide sequence of atranscript variant. Table XXV shows the alignment of the transcriptvariant 158P3D2 var2 with nucleic acid sequence of 158P3D2 var1. TableXXVI lays out amino acid translation of the transcript variant 158P3D2var2 for the identified reading frame orientation. Table XXVII displaysalignments of the amino acid sequence encoded by the transcript variant158P3D2 var2 with that of 158P3D2 var1.

Example 6 Single Nucleotide Polymorphisms of 158P3D2

[0469] Single Nucleotide Polymorphism (SNP) is a single base pairvariation in nucleotide sequences. At a specific point of the genome,there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A.Genotype refers to the base pair make-up of one or more spots in thegenome of an individual, while haplotype refers to base pair make-up ofmore than one varied spots on the same DNA molecule (chromosome inhigher organism). SNPs that occur on a cDNA are called cSNPs. ThesecSNPs may change amino acids of the protein encoded by the gene and thuschange the functions of the protein. Some SNPs cause inherited diseasesand some others contribute to quantitative variations in phenotype andreactions to environmental factors including diet and drugs amongindividuals. Therefore, SNPs and/or combinations of alleles (calledhaplotypes) have many applications including diagnosis of inheriteddiseases, determination of drug reactions and dosage, identification ofgenes responsible for diseases and discovery of genetic relationshipbetween individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNPanalysis to dissect human traits,” Curr. Opin. Neurobiol. October 2001;11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility toadverse drug reactions,” Trends Pharmacol. Sci. June 2001;22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The useof single nucleotide polymorphisms in the isolation of common diseasegenes,” Pharmacogenonics. February 2000; 1(1):39-47; R. Judson, J. C.Stephens and A. Windemuth, “The predictive power of haplotypes inclinical response,” Pharmacogenomics. February 2000; 1(1):15-26).

[0470] SNPs are identified by a variety of art-accepted methods (P.Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab.October-November 2001; 20(9): 18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. July 1998; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. February 2001;47(2): 164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.December 2000; 5(4):329-340).

[0471] SNPs are identified by directly sequencing cDNA clones of theinvention and by comparing the sequences with public and proprietarysequences. By comparing these cDNA clones with high quality proprietaryor public sequences, seven SNPs were identified and two of them arelinked (a deletion and a substitution). The transcripts or proteins withalternative alleles were designated as variants 158P3D2 v.3, v.4, v.5,v.6, v.7 and v.8. FIG. 10 shows the schematic alignment of thenucleotide variants. FIG. 11 shows the schematic alignment of proteinvariants, corresponding to nucleotide variants. Nucleotide variants thatcode for the same amino acid sequence as variant 1 are not shown in FIG.11. These alleles of the SNPs, though shown separately here, can occurin different combinations (haplotypes) and in different transcriptvariants that contain the sequence context.

Example 7 Production of Recombinant 158P3D2 in Prokaryotic Systems

[0472] To express recombinant 158P3D2 and 158P3D2 variants inprokaryotic cells, the full or partial length 158P3D2 and 158P3D2variant cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 158P3D2 or 158P3D2 variants are expressed in theseconstructs, amino acids 1 to 328 of 158P3D2 (variant 1), amino acids1-236 of variant 2a, amino acids 1-181 of variant 2b, amino acids 1-328of variant 3, amino acids 1-328 of variant 4, amino acids 1-178 ofvariant 5a, amino acids 1-181 of variant 5b; or any 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more contiguous amino acids from 158P3D2, variants, or analogsthereof.

[0473] A. In vitro Transcription and Translation Constructs:

[0474] pCRII: To generate 158P3D2 sense and anti-sense RNA probes forRNA in situ investigations, pCRII constructs (Invitrogen, CarlsbadCalif.) are generated encoding either all or fragments of the 158P3D2cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert todrive the transcription of 158P3D2 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 158P3D2 at the RNA level. Transcribed 158P3D2 RNArepresenting the cDNA amino acid coding region of the 158P3D2 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize158P3D2 protein.

[0475] B. Bacterial Constructs:

[0476] pGEX Constructs: To generate recombinant 158P3D2 proteins inbacteria that are fused to the Glutathione S-transferase (GST) protein,all or parts of the T-fusion vector of the pGEX family (AmershamPharmacia Biotech, Piscataway, N.J.). These constructs allow controlledexpression of recombinant 158P3D2 protein sequences with GST fused atthe amino-terminus and a six histidine epitope (6×His) at thecarboxyl-terminus. The GST and 6×His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6×His tag is generated by adding 6histidine codons to the cloning primer at the 3′ end, e.g., of the openreading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 158P3D2-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

[0477] pMAL Constructs: To generate, in bacteria, recombinant 158P3D2proteins that are fused to maltose-binding protein (MBP), all or partsof the 158P3D2 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 158P3D2 protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 158P3D2. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

[0478] pET Constructs: To express 158P3D2 in bacterial cells, all orparts of the 158P3D2 cDNA protein coding sequence are cloned into thepET family of vectors (Novagen, Madison, Wis.). These vectors allowtightly controlled expression of recombinant 158P3D2 protein in bacteriawith and without fusion to proteins that enhance solubility, such asNusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™that aid purification and detection of the recombinant protein. Forexample, constructs are made utilizing pET NusA fusion system 43.1 suchthat regions of the 158P3D2 protein are expressed as amino-terminalfusions to NusA. In one embodiment, a NusA-fusion protein encompassingamino acids 412-328 of 158P3D2 with a C-terminal 6×His tag was expressedin E. Coli, purified by metal chelate affinity chromatography, and usedas an immunogen for generation of antibodies.

[0479] C. Yeast Constructs:

[0480] pESC Constructs: To express 158P3D2 in the yeast speciesSaccharomyces cerevisiae for generation of recombinant protein andfunctional studies, all or parts of the 158P3D2 cDNA protein codingsequence are cloned into the pESC family of vectors each of whichcontain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3(Stratagene, La Jolla, Calif.). These vectors allow controlledexpression from the same plasmid of up to 2 different genes or clonedsequences containing either Flag™ or Myc epitope tags in the same yeastcell. This system is useful to confirm protein-protein interactions of158P3D2. In addition, expression in yeast yields similarpost-translational modifications, such as glycosylations andphosphorylations, that are found when expressed in eukaryotic cells.

[0481] pESP Constructs: To express 158P3D2 in the yeast speciesSaccharomyces pombe, all or parts of the 158P3D2 cDNA protein codingsequence are cloned into the pESP family of vectors. These vectors allowcontrolled high level of expression of a 158P3D2 protein sequence thatis fused at either the amino terminus or at the carboxyl terminus to GSTwhich aids purification of the recombinant protein. A Flag™ epitope tagallows detection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 158P3D2 in Eukaryotic Systems

[0482] A. Mammalian Constructs:

[0483] To express recombinant 158P3D2 in eukaryotic cells, the full orpartial length 158P3D2 cDNA sequences were cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 158P3D2 were expressed in these constructs, aminoacids 1 to 328, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acidsfrom 158P3D2, variants, or analogs thereof. In certain embodiments aregion of 158P3D2 was expressed that encodes an amino acid not sharedamongst at least variants.

[0484] The constructs were transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-158P3D2 polyclonal serum, described herein.

[0485] pcDNA4/HisMax Constructs: To express 158P3D2 in mammalian cells,a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

[0486] pcDNA3.1/MycHis Constructs: To express 158P3D2 in mammaliancells, a 158P3D2 ORF, or portions thereof, of 158P3D2 with a consensusKozak translation initiation site was cloned into pcDNA3.1/MycHisVersion A (Invitrogen, Carlsbad, Calif.). Protein expression is drivenfrom the cytomegalovirus (CMV) promoter. The recombinant proteins havethe myc epitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. FIG. 19 shows expression of 158P3D2.pcDNA3.1/mychis intransiently transfected 293T cells.

[0487] pcDNA3.1/CT-GFP-TOPO Construct: To express 158P3D2 in mammaliancells and to allow detection of the recombinant proteins usingfluorescence, a 158P3D2 ORF, or portions thereof, with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, CA). Protein expression is driven from the cytomegalovirus(CMV) promoter. The recombinant proteins have the Green FluorescentProtein (GFP) fused to the carboxyl-terminus facilitating non-invasive,in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPOvector also contains the bovine growth hormone (BGH) polyadenylationsignal and transcription termination sequence to enhance mRNA stabilityalong with the SV40 origin for episomal replication and simple vectorrescue in cell lines expressing the large T antigen. The Neomycinresistance gene allows for selection of mammalian cells that express theprotein, and the ampicillin resistance gene and ColE1 origin permitsselection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of a 158P3D2 protein.

[0488] PAPtag: A 158P3D2 ORF, or portions thereof, is cloned intopAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates analkaline phosphatase fusion at the carboxyl-terminus of a 158P3D2protein while fusing the IgGK signal sequence to the amino-terminus.Constructs are also generated in which alkaline phosphatase with anamino-terminal IgGK signal sequence is fused to the amino-terminus of a158P3D2 protein. The resulting recombinant 158P3D2 proteins areoptimized for secretion into the media of transfected mammalian cellsand can be used to identify proteins such as ligands or receptors thatinteract with 158P3D2 proteins. Protein expression is driven from theCMV promoter and the recombinant proteins also contain myc and 6×Hisepitopes fused at the carboxyl-terminus that facilitates detection andpurification. The Zeocin resistance gene present in the vector allowsfor selection of mammalian cells expressing the recombinant protein andthe ampicillin resistance gene permits selection of the plasmid in E.coli. ptag5: A 158P3D2 ORF, or portions thereof, is cloned into pTag-5.This vector is similar to pAPtag but without the alkaline phosphatasefusion. This construct generates 158P3D2 protein with an amino-terminalIgGK signal sequence and myc and 6×His epitope tags at thecarboxyl-terminus that facilitate detection and affinity purification.The resulting recombinant 158P3D2 protein is optimized for secretioninto the media of transfected mammalian cells, and is used as immunogenor ligand to identify proteins such as ligands or receptors thatinteract with the 158P3D2 proteins. Protein expression is driven fromthe CMV promoter. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

[0489] PsecFc: A 158P3D2 ORF, or portions thereof, is also cloned intopsecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, California). This construct generates an IgG1 Fc fusion atthe carboxyl-terminus of the 158P3D2 proteins, while fusing the IgGKsignal sequence to N-terminus. 158P3D2 fusions utilizing the murine IgG1Fc region are also used. The resulting recombinant 158P3D2 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 158P3D2 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

[0490] pSRα Constructs: To generate mammalian cell lines that express158P3D2 constitutively, 158P3D2 ORF, or portions thereof, of 158P3D2 arecloned into pSRα constructs. Amphotropic and ecotropic retroviruses aregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 158P3D2,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

[0491] Additional pSRα constructs are made that fuse an epitope tag suchas the FLAG™ tag to the carboxyl-terminus of 158P3D2 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ is added to cloning primer at the 3′end of the ORF. Additional pSRα constructs are made to produce bothamino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteinsof the full-length 158P3D2 proteins.

[0492] Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 158P3D2. High virus titerleading to high level expression of 158P3D2 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 158P3D2 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 158P3D2coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-I cells.

[0493] Regulated Expression Systems: To control expression of 158P3D2 inmammalian cells, coding sequences of 158P3D2, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant158P3D2. These vectors are thereafter used to control expression of158P3D2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

[0494] B. Baculovirus Expression Systems

[0495] To generate recombinant 158P3D2 proteins in a baculovirusexpression system, 158P3D2 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-158P3D2 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

[0496] Recombinant 158P3D2 protein is then generated by infection ofHighFive insect cells (Invitrogen) with purified baculovirus.Recombinant 158P3D2 protein can be detected using anti-158P3D2 oranti-His-tag antibody. 158P3D2 protein can be purified and used invarious cell-based assays or as immunogen to generate polyclonal andmonoclonal antibodies specific for 158P3D2.

Example 9 Antigenicity Profiles and Secondary Structure

[0497]FIG. 5A, FIG. 6A, FIG. 7A, FIG. 8A, and FIG. 9A depict graphicallyfive amino acid profiles of the 158P3D2 variant 1 amino acid sequence;FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, and FIG. 9B depict graphically fiveamino acid profiles of the 158P3D2 variant 2A amino acid sequence, andFIG. 5C, FIG. 6C, FIG. 7C, FIG. 8C, and FIG. 9C depict graphically fiveamino acid profiles of the 158P3D2 variant 5A amino acid sequence, eachassessment available by accessing the ProtScale website (URLwww.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biologyserver.

[0498] These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R.,1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6,Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., andPonnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9,Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); andoptionally others available in the art, such as on the ProtScalewebsite, were used to identify antigenic regions of the 158P3D2 protein.Each of the above amino acid profiles of 158P3D2 were generated usingthe following ProtScale parameters for analysis: 1) A window size of 9;2) 100% weight of the window edges compared to the window center; and,3) amino acid profile values normalized to lie between 0 and 1.

[0499] Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

[0500] Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profilesdetermine stretches of amino acids (i.e., values greater than 0.5 on theBeta-turn profile and the Average Flexibility profile) that are notconstrained in secondary structures such as beta sheets and alphahelices. Such regions are also more likely to be exposed on the proteinand thus accessible to immune recognition, such as by antibodies.

[0501] Antigenic sequences of the 158P3D2 protein and of the variantproteins indicated, e.g., by the profiles set forth in FIGS. 5A-C, FIGS.6A-C, FIGS. 7A-C, FIGS. 8A-C, and/or FIGS. 9A-C are used to prepareimmunogens, either peptides or nucleic acids that encode them, togenerate therapeutic and diagnostic anti-158P3D2antibodies. Theimmunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 158P3D2 protein or of 158P3D2 variants. In particular,peptide immunogens of the invention can comprise, a peptide region of atleast 5 amino acids of FIG. 2 in any whole number increment up to 328that includes an amino acid position having a value greater than 0.5 inthe Hydrophilicity profile of FIG. 5; a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to 328 thatincludes an amino acid position having a value less than 0.5 in theHydropathicity profile of FIG. 6; a peptide region of at least 5 aminoacids of FIG. 2 in any whole number increment up to 328 that includes anamino acid position having a value greater than 0.5 in the PercentAccessible Residues profile of FIG. 7; a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to 328 thatincludes an amino acid position having a value greater than 0.5 in theAverage Flexibility profile on FIG. 8; and, a peptide region of at least5 amino acids of FIG. 2 in any whole number increment up to 328 thatincludes an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9. Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

[0502] All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

[0503] The secondary structure of 158P3D2 variant 1 and variants 2a and5a, namely the predicted presence and location of alpha helices,extended strands, and random coils, is predicted from the primary aminoacid sequence using the HNN—Hierarchical Neural Network method(Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).The analysis indicates that 158P3D2 variant 1 is composed 32.93% alphahelix, 18.29% extended strand, and 48.78% random coil (FIG. 12A),variant 2a is composed of 25.85% alpha helix, 18.22% extended strand,and 55.93% random coil (FIG. 12B), and variant 5a is composed of 9.55%alpha helix, 26.40% extended strand, and 64.04% random coil (FIG. 12C).

[0504] Analysis for the potential presence of transmembrane domains in158P3D2 variant 1 was carried out using a variety of transmembraneprediction algorithms accessed from the ExPasy molecular biology server(http://www.expasy.ch/tools/). The programs predict the presence of asingle transmembrane domain in 15.8P3D2 variant 1. Shown graphically inFIGS. 12D and 12E are the results of analysis using the TMpred (FIG.12D) and TMHMM (FIG. 12E) prediction programs depicting the location ofthe transmembrane domain. The results of each program, namely the aminoacids encoding the transmembrane domain are summarized in Table XXII.Variants 2b, 3, 4, and 5b, also contain the amino acids predicted toencode the transmembrane domain. No transmembrane domains are predictedin variants 2a and 5a.

Example 10 Generation of 158P3D2 Polyclonal Antibodies

[0505] Polyclonal antibodies can be raised in a mammal, for example, byone or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. In addition to immunizing with the full length 158P3D2protein, computer algorithms are employed in design of immunogens that,based on amino acid sequence analysis contain characteristics of beingantigenic and available for recognition by the immune system of theimmunized host (see Example 9): Such regions would be predicted to behydrophilic, flexible, in beta-turn conformations, and be exposed on thesurface of the protein (see, e.g., FIGS. 5A-C, FIGS. 6A-C, FIGS. 7A-C,FIGS. 8A-C, or FIGS. 9A-C for amino acid profiles that indicate suchregions of 158P3D2 and variants).

[0506] For example, 158P3D2 recombinant bacterial fusion proteins orpeptides containing hydrophilic, flexible, beta-turn regions of the158P3D2, such as regions amino terminal to the predicted transmembranedomain of variant 1 (predicted to be extracellular), are used asantigens to generate polyclonal antibodies in New Zealand White rabbits.For example, such regions include, but are not limited to, amino acids1-25, amino acids 37-54, amino acids 60-73, and amino acids 187-225. Itis useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include, but are not limited to, keyhole limpet hemocyanin(KLH), serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. In one embodiment, a peptide encoding amino acids 200-225 of158P3D2 is conjugated to KLH and used to immunize the rabbit.Alternatively the immunizing agent may include all or portions of the158P3D2 protein, analogs or fusion proteins thereof. For example, the158P3D2 amino acid sequence can be fused using recombinant DNAtechniques to any one of a variety of fusion protein partners that arewell known in the art, such as glutathione-S-transferase (GST) and HIStagged fusion proteins. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

[0507] In one embodiment, a GST-fusion protein encoding the predictedextracellular domain, amino acids 1-291, is produced and purified andused as immunogen. Other recombinant bacterial fusion proteins that maybe employed include maltose binding protein, LacZ, thioredoxin, NusA, oran immunoglobulin constant region (see Example 7 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al.eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle,N., and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).

[0508] In addition to bacterial derived fusion proteins, mammalianexpressed protein antigens are also used. These antigens are expressedfrom mammalian expression vectors such as the Tag5 and Fc-fusion vectors(see Example 8), and retain post-translational modifications such asglycosylations found in native protein. 10. In one embodiment, aminoacids 185-225 is cloned into the TagS mammalian secretion vector. Inanother embodiment, the predicted extracellular domain, amino acids1-291 is cloned into the TagS expression vector. The recombinantproteins are purified by metal chelate chromatography from tissueculture supernatants of 293T cells stably expressing the recombinantvector. The purified Tag5 158P3D2 proteins are then individually used asimmunogens.

[0509] During the immunization protocol, it is useful to mix or emulsifythe antigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

[0510] In a typical protocol, rabbits are initially immunizedsubcutaneously with up to 200 μg, typically 100-200 μg, of fusionprotein or peptide conjugated to KLH mixed in complete Freund's adjuvant(CFA). Rabbits are then injected subcutaneously every two weeks with upto 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund'sadjuvant (IFA). Test bleeds are taken approximately 7-10 days followingeach immunization and used to monitor the titer of the antiserum byELISA.

[0511] To test reactivity and specificity of immune serum, such as therabbit serum derived from immunization with TagS 158P3D2 encoding aminoacids 1-291, the full-length 158P3D2 cDNA is cloned into pcDNA 3.1myc-his expression vector (Invitrogen, see Example 7). Aftertransfection of the constructs into 293T cells, cell lysates are probedwith the anti-158P3D2 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 158P3D2 protein using the Western blot technique. Shown inFIG. 19 is expression of Myc His tagged 158P3D2 protein in 293T cells asdetected by Western blot with anti-His antibody. The immune serum isthen tested by the Western blot technique against 293T-158P3D2 cells. Inaddition, the immune serum is tested by fluorescence microscopy, flowcytometry and immunoprecipitation against 293T and other recombinant158P3D2-expressing cells to determine specific recognition of nativeprotein. Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express 158P3D2are also carried out to test reactivity and specificity.

[0512] Anti-serum from rabbits immunized with 158P3D2 fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-158P3D2 fusion protein encoding amino acids 1-291 is firstpurified by passage over a column of GST protein covalently coupled toAffiGel matrix (BioRad, Hercules, Calif.). The antiserum is thenaffinity purified by passage over a column composed of a MBP-fusionprotein also encoding amino acids 1-291 covalently coupled to Affigelmatrix. The serum is then further purified by protein G affinitychromatography to isolate the IgG fraction. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 11 Generation of 158P3D2 Monoclonal Antibodies (mAbs)

[0513] In one embodiment, therapeutic mAbs to 158P3D2 comprise thosethat react with epitopes of the protein that would disrupt or modulatethe biological function of 158P3D2, for example those that would disruptits interaction with ligands and binding partners. Therapeutic mAbs alsocomprise those that specifically bind epitopes of 158P3D2 exposed on thecell surface and thus are useful in targeting mAb-toxin conjugates.Immunogens for generation of such mAbs include those designed to encodeor contain the entire 158P3D2 protein, regions of the 158P3D2 proteinpredicted to be antigenic from computer analysis of the amino acidsequence (see, e.g., FIGS. 5A-C, FIGS. 6A-C, FIGS. 7A-C, FIGS. 8A-C, orFIGS. 9A-C, and Example 9) such as regions in the extracellular domainof variant 1. Immunogens include peptides, recombinant bacterialproteins, and mammalian expressed Tag 5 proteins and human and murineIgG FC fusion proteins. In addition, cells expressing high levels of158P3D2, such as 293T-158P3D2 or 300.19-158P3D2 murine Pre-B cells, areused to immunize mice.

[0514] To generate mAbs to 158P3D2, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 158P3D2-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding 158P3D2 sequence is used to immunize mice bydirect injection of the plasmid DNA. For example, amino acids 1-291 iscloned into the Tag5 mammalian secretion vector and the recombinantvector is used as immunogen. In another example the same amino acids arecloned into an Fc-fusion secretion vector in which the 158P3D2 sequenceis fused at the amino-terminus to an IgK leader sequence and at thecarboxyl-terminus to the coding sequence of the human or murine IgG Fcregion. This recombinant vector is then used as immunogen. The plasmidimmunization protocols are used in combination with purified proteinsexpressed from the same vector and with cells expressing 158P3D2.

[0515] During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

[0516] In one embodiment for generating 158P3D2 monoclonal antibodies, aTag5-158P3D2 antigen encoding amino acids 1-291, the predictedextracellular domain, is expressed and purified from stably transfected293T cells. Balb C mice are initially immunized intraperitoneally with25 μg of the Tag5-158P3D2 protein mixed in complete Freund's adjuvant.Mice are subsequently immunized every two weeks with 25 μg of theantigen mixed in incomplete Freund's adjuvant for a total of threeimmunizations. ELISA using the Tag5 antigen determines the titer ofserum from immunized mice. Reactivity and specificity of serum to fulllength 158P3D2 protein is monitored by Western blotting,immunoprecipitation and flow cytometry using 293T cells transfected withan expression vector encoding the 158P3D2 cDNA (see e.g., Example 8).Other recombinant 158P3D2-expressing cells or cells endogenouslyexpressing 158P3D2 are also used. Mice showing the strongest reactivityare rested and given a final injection of Tag5 antigen in PBS and thensacrificed four days later. The spleens of the sacrificed mice areharvested and fused to SPO/2 myeloma cells using standard procedures(Harlow and Lane, 1988). Supernatants from HAT selected growth wells arescreened by ELISA, Western blot, immunoprecipitation, fluorescentmicroscopy, and flow cytometry to identify 158P3D2 specificantibody-producing clones.

[0517] Monoclonal antibodies are also derived that react only withspecific 158P3D2 variants, such as variants 2a and 5a. To this end,immunogens are designed to encode amino acid regions specific to therespective variant. For example, a Tag5 immunogen is encoding aminoacids 1-236 of variant 2a is produced, purified, and used to immunizemice to generate hybridomas. In another example, a Tag5 immunogenencoding amino acids 130-178 of variant 5a is produced, purified, andused as immunogen. Monoclonal antibodies raised to these immunogens arethen screened for reactivity to cells expressing the respective variantsbut not to other 158P3D2 variants. These strategies for raising 158P3D2variant specific monoclonal antibodies are also applied to polyclonalreagents described in Example 10.

[0518] The binding affinity of a 158P3D2 monoclonal antibody isdetermined using standard technologies. Affinity measurements quantifythe strength of antibody to epitope binding and are used to help definewhich 158P3D2 monoclonal antibodies preferred for diagnostic ortherapeutic use, as appreciated by one of skill in the art. The BIAcoresystem (Uppsala, Sweden) is a preferred method for determining bindingaffinity. The BIAcore system uses surface plasmon resonance (SPR,Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998,Methods in Enzymology 295: 268) to monitor biomolecular interactions inreal time. BIAcore analysis conveniently generates association rateconstants, dissociation rate constants, equilibrium dissociationconstants, and affinity constants.

Example 12 HLA Class I and Class II Binding Assays

[0519] HLA class I and class II binding assays using purified HLAmolecules are performed in accordance with disclosed protocols (e.g.,PCT publications WO 94/20127 and WO 94/03205; Sidney et al., CurrentProtocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol.154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly,purified MHC molecules (5 to 500 nM) are incubated with variousunlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probepeptides as described. Following incubation, MHC-peptide complexes areseparated from free peptide by gel filtration and the fraction ofpeptide bound is determined. Typically, in preliminary experiments, eachMHC preparation is titered in the presence of fixed amounts ofradiolabeled peptides to determine the concentration of HLA moleculesnecessary to bind 10-20% of the total radioactivity. All subsequentinhibition and direct binding assays are performed using these HLAconcentrations.

[0520] Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], themeasured IC₅₀ values are reasonable approximations of the true K_(D)values. Peptide inhibitors are typically tested at concentrationsranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to fourcompletely independent experiments. To allow comparison of the dataobtained in different experiments, a relative binding figure iscalculated for each peptide by dividing the IC₅₀ of a positive controlfor inhibition by the IC₅₀ for each tested peptide (typically unlabeledversions of the radiolabeled probe peptide). For database purposes, andinter-experiment comparisons, relative binding values are compiled.These values can subsequently be converted back into IC₅₀ nM values bydividing the IC₅₀ nM of the positive controls for inhibition by therelative binding of the peptide of interest. This method of datacompilation is accurate and consistent for comparing peptides that havebeen tested on different days, or with different lots of purified MHC.

[0521] Binding assays as outlined above may be used to analyze HLAsupermotif and/or HLA motif-bearing peptides.

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

[0522] HLA vaccine compositions of the invention can include multipleepitopes. The multiple epitopes can comprise multiple HLA supermotifs ormotifs to achieve broad population coverage. This example illustratesthe identification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

[0523] Computer Searches and Algorithms for Identification of Supermotifand/or Motif-Bearing Epitopes

[0524] The searches performed to identify the motif-bearing peptidesequences in Example 9 and Tables V-XIX employ the protein sequence datafrom the gene product of 158P3D2 set forth in FIGS. 2 and 3.

[0525] Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 158P3D2protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

[0526] Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

[0527] where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

[0528] The method of derivation of specific algorithm coefficients hasbeen described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997;(see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood etal., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions,anchor and non-anchor alike, the geometric mean of the average relativebinding (ARB) of all peptides carrying j is calculated relative to theremainder of the group, and used as the estimate of j_(i). For Class IIpeptides, if multiple alignments are possible, only the highest scoringalignment is utilized, following an iterative procedure. To calculate analgorithm score of a given peptide in a test set, the ARB valuescorresponding to the sequence of the peptide are multiplied. If thisproduct exceeds a chosen threshold, the peptide is predicted to bind.Appropriate thresholds are chosen as a function of the degree ofstringency of prediction desired.

[0529] Selection of HLA-A2 Supertype Cross-Reactive Peptides

[0530] Protein sequences from 158P3D2 are scanned utilizing motifidentification software, to identify 8-, 9- 10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

[0531] These peptides are then tested for the capacity to bind toadditional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802).Peptides that bind to at least three of the five A2-supertype allelestested are typically deemed A2-supertype cross-reactive binders.Preferred peptides bind at an affinity equal to or less than 500 nM tothree or more HLA-A2 supertype molecules.

[0532] Selection of HLA-A3 Supermotif-Bearing Epitopes

[0533] The 158P3D2 protein sequence(s) scanned above is also examinedfor the presence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A* 1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of <500 nM, often <200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

[0534] Selection of HLA-B7 Supermotif Bearing Epitopes

[0535] The 158P3D2 protein(s) scanned above is also analyzed for thepresence of 8-, 9-10-, or 1 1-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of <500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

[0536] Selection of A1 and A24 Motif-Bearing Epitopes

[0537] To further increase population coverage, HLA-A1 and -A24 epitopescan also be incorporated into vaccine compositions. An analysis of the158P3D2 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

[0538] High affinity and/or cross-reactive binding epitopes that bearother motif and/or supermotifs arc identified using analogousmethodology.

Example 14 Confirmation of Immunogenicity

[0539] Cross-reactive candidate CTL A2-supermotif-bearing peptides thatare identified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

[0540] Target Cell Lines for Cellular Screening:

[0541] The 0.221A2.1 cell line, produced by transferring the HLA-A2.1gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line721.221, is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

[0542] Primary CTL Induction Cultures:

[0543] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with30 μg/ml DNAse, washed twice and resuspended in complete medium(RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodiumpyruvate, L-glutamine and penicillin/streptomycin). The monocytes arepurified by plating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at37° C., the non-adherent cells are removed by gently shaking the platesand aspirating the supernatants. The wells are washed a total of threetimes with 3 ml RPMI to remove most of the non-adherent and looselyadherent cells. Three ml of complete medium containing 50 ng/ml ofGM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is addedto the DCs on day 6 at 75 ng/ml and the cells are used for CTL inductioncultures on day 7.

[0544] Induction of CTL with DC and Peptide: CD8+T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶cells/ml. The magnetic beadsare washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4×with PBS/AB serum to remove thenonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

[0545] Setting up induction cultures: 0.25 ml cytokine-generated DC (at1×10⁵ cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

[0546] Restimulation of the induction cultures with peptide-pulsedadherent cells: Seven and fourteen days after the primary induction, thecells are restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/mil and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25milRPMI/5%AB per well for 2 hours at 37° C. Peptide solution from each wellis aspirated and the wells are washed once with RPMI. Most of the mediais aspirated from the induction cultures (CD8+cells) and brought to 0.5ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

[0547] Measurement of CTL Lytic Activity by ⁵¹Cr Release.

[0548] Seven days after the second restimulation, cytotoxicity isdetermined in a standard (5 hr) ⁵¹Cr release assay by assayingindividual wells at a single E:T. Peptide-pulsed targets are prepared byincubating the cells with 10 μg/ml peptide overnight at 37° C.

[0549] Adherent target cells are removed from culture flasks withtrypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹Cr sodiumchromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled targetcells are resuspended at 106 per ml and diluted 1:10 with K562 cells ata concentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

[0550] Maximum and spontaneous release are determined by incubating thelabeled targets with 1% Triton X-100 and media alone, respectively. Apositive culture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

[0551] In situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

[0552] Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH 8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

[0553] Recombinant human IFN-gamma is added to the standard wellsstarting at 400 μg or 1200 μg/100 microliter/well and the plateincubated for two hours at 37° C. The plates are washed and 100 μl ofbiotinylated mouse anti-human IFN-gamma monoclonal antibody (2microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2hours at room temperature. After washing again, 100 microliterHRP-streptavidin (1:4000) are added and the plates incubated for onehour at room temperature. The plates are then washed 6×with wash buffer,100 microliter/well developing solution (TMB 1:1) are added, and theplates allowed to develop for 5-15 minutes. The reaction is stopped with50 microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

[0554] CTL Expansion.

[0555] Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

[0556] Cultures are expanded in the absence of anti-CD3⁺ as follows.Those cultures that demonstrate specific lytic activity against peptideand endogenous targets are selected and 5×10⁴ CD8⁺ cells are added to aT25 flask containing the following: 1×10⁶ autologous PBMC per ml whichhave been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C.and irradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essentialAA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

[0557] Immunogenicity of A2 Supermotif-Bearing Peptides

[0558] A2-supermotif cross-reactive binding peptides are tested in thecellular assay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

[0559] Immunogenicity can also be confirmed using PBMCs isolated frompatients bearing a tumor that expresses 158P3D2. Briefly, PBMCs areisolated from patients, re-stimulated with peptide-pulsed monocytes andassayed for the ability to recognize peptide-pulsed target cells as wellas transfected cells endogenously expressing the antigen.

[0560] Evaluation of A*03/A11 Immunogenicity

[0561] HLA-A3 supermotif-bearing cross-reactive binding peptides arealso evaluated for immunogenicity using methodology analogous for thatused to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

[0562] Evaluation of B7 Immunogenicity

[0563] Immunogenicity screening of the B7-supertype cross-reactivebinding peptides identified as set forth herein are confirmed in amanner analogous to the confirmation of A2-and A3-supermotif-bearingpeptides.

[0564] Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24etc. are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

[0565] HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

[0566] Analoging at Primary Anchor Residues

[0567] Peptide engineering strategies are implemented to furtherincrease the cross-reactivity of the epitopes. For example, the mainanchors of A2-supermotif-bearing peptides are altered, for example, tointroduce a preferred L, I, V, or M at position 2, and I or V at theC-terminus.

[0568] To analyze the cross-reactivity of the analog peptides, eachengineered analog is initially tested for binding to the prototype A2supertype allele A*0201, then, if A*0201 binding capacity is maintained,for A2-supertype cross-reactivity.

[0569] Alternatively, a peptide is confirmed as binding one or allsupertype members and then analoged to modulate binding affinity to anyone (or more) of the supertype members to add population coverage.

[0570] The selection of analogs for immunogenicity in a cellularscreening analysis is typically further restricted by the capacity ofthe parent wild type (WT) peptide to bind at least weakly, i.e., bind atan IC₅₀ of 5000 nM or less, to three of more A2 supertype alleles. Therationale for this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

[0571] In the cellular screening of these peptide analogs, it isimportant to confirm that analog-specific CTLs are also able torecognize the wild-type peptide and, when possible, target cells thatendogenously express the epitope.

[0572] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

[0573] Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

[0574] The analog peptides are then tested for the ability to bind A*03and A*11 (prototype A3 supertype alleles). Those peptides thatdemonstrate ≦500 nM binding capacity are then confirmed as havingA3-supertype cross-reactivity.

[0575] Similarly to the A2- and A3-motif bearing peptides, peptidesbinding 3 or more B7-supertype alleles can be improved, where possible,to achieve increased cross-reactive binding or greater binding affinityor binding half life. B7 supermotif-bearing peptides are, for example,engineered to possess a preferred residue (V, I, L, or F) at theC-terminal primary anchor position, as demonstrated by Sidney et al. (J.Immunol. 157:3480-3490, 1996).

[0576] Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

[0577] The analog peptides are then be confirmed for immunogenicity,typically in a cellular screening assay. Again, it is generallyimportant to demonstrate that analog-specific CTLs are also able torecognize the wild-type peptide and, when possible, targets thatendogenously express the epitope.

[0578] Analoging at Secondary Anchor Residues

[0579] Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

[0580] Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with158P3D2-expressing tumors.

[0581] Other Analoging Strategies

[0582] Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

[0583] Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 158P3D2-Derived Sequenceswith HLA-DR Binding Motifs

[0584] Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

[0585] Selection of HLA-DR-Supermotif-Bearing Epitopes.

[0586] To identify 158P3D2-derived, HLA class II HTL epitopes, a 158P3D2antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

[0587] Protocols for predicting peptide binding to DR molecules havebeen developed (Southwood et al., J. Immunol. 160:3363-3373, 1998).These protocols, specific for individual DR molecules, allow thescoring, and ranking, of 9-mer core regions. Each protocol not onlyscores peptide sequences for the presence of DR-supermotif primaryanchors (i.e., at position 1 and position 6) within a 9-mer core, butadditionally evaluates sequences for the presence of secondary anchors.Using allele-specific selection tables (see, e.g. Southwood et al.,ibid.), it has been found that these protocols efficiently selectpeptide sequences with a high probability of binding a particular DRmolecule. Additionally, it has been found that performing theseprotocols in tandem, specifically those for DR1, DR4w4, and DR7, canefficiently select DR cross-reactive peptides.

[0588] The 158P3D2-derived peptides identified above are tested fortheir binding capacity for various common HLA-DR molecules. All peptidesare initially tested for binding to the DR molecules in the primarypanel: DR1, DR4w4, and DR7. Peptides binding at least two of these threeDR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19,and DR9 molecules in secondary assays. Finally, peptides binding atleast two of the four secondary panel DR molecules, and thuscumulatively at least four of seven different DR molecules, are screenedfor binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays.Peptides binding at least seven of the ten DR molecules comprising theprimary, secondary, and tertiary screening assays are consideredcross-reactive DR binders. 158P3D2-derived peptides found to bind commonHLA-DR alleles are of particular interest.

[0589] Selection of DR3 Motif Peptides

[0590] Because HLA-DR3 is an allele that is prevalent in Caucasian,Black, and Hispanic populations, DR3 binding capacity is a relevantcriterion in the selection of HTL epitopes. Thus, peptides shown to becandidates may also be assayed for their DR3 binding capacity. However,in view of the binding specificity of the DR3 motif, peptides bindingonly to DR3 can also be considered as candidates for inclusion in avaccine formulation.

[0591] To efficiently identify peptides that bind DR3, target 158P3D2antigens are analyzed for sequences carrying one of the two DR3-specificbinding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748,1994). The corresponding peptides are then synthesized and confirmed ashaving the ability to bind DR3 with an affinity of 1 μM or better, i.e.,less than 1 μM. Peptides are found that meet this binding criterion andqualify as HLA class II high affinity binders.

[0592] DR3 binding epitopes identified in this manner are included invaccine compositions with DR supermotif-bearing peptide epitopes.

[0593] Similarly to the case of HLA class I motif-bearing peptides, theclass II motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 158P3D2-Derived HTL Epitopes

[0594] This example determines immunogenic DR supermotif- and DR3motif-bearing epitopes among those identified using the methodology setforth herein.

[0595] Immunogenicity of HTL epitopes are confirmed in a manneranalogous to the determination of immunogenicity of CTL epitopes, byassessing the ability to stimulate HTL responses and/or by usingappropriate transgenic mouse models. Immunogenicity is determined byscreening for: 1.) in vitro primary induction using normal PBMC or 2.)recall responses from patients who have 158P3D2-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

[0596] This example illustrates the assessment of the breadth ofpopulation coverage of a vaccine composition comprised of multipleepitopes comprising multiple supermotifs and/or motifs.

[0597] In order to analyze population coverage, gene frequencies of HLAalleles are determined. Gene frequencies for each HLA allele arecalculated from antigen or allele frequencies utilizing the binomialdistribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., HumanImmunol. 45:79-93, 1996). To obtain overall phenotypic frequencies,cumulative gene frequencies are calculated, and the cumulative antigenfrequencies derived by the use of the inverse formula [af=1-(1-Cgf)²].

[0598] Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

[0599] Population coverage achieved by combining the A2-, A3- andB7-supertypes is approximately 86% in five major ethnic groups. Coveragemay be extended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

[0600] Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin.Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeldet al., J. Immunol. 159:1648, 1997) have shown that highlycross-reactive binding peptides are almost always recognized asepitopes. The use of highly cross-reactive binding peptides is animportant selection criterion in identifying candidate epitopes forinclusion in a vaccine that is immunogenic in a diverse population.

[0601] With a sufficient number of epitopes (as disclosed herein andfrom the art), an average population coverage is predicted to be greaterthan 95% in each of five major ethnic populations. The game theory MonteCarlo simulation analysis, which is known in the art (see e.g., Osborne,M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), canbe used to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition of Endogenously Processed Antigens afterPriming

[0602] This example confirms that CTL induced by native or analogedpeptide epitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

[0603] Effector cells isolated from transgenic mice that are immunizedwith peptide epitopes, for example HLA-A2 supermotif-bearing epitopes,are re-stimulated in vitro using peptide-coated stimulator cells. Sixdays later, effector cells are assayed for cytotoxicity and the celllines that contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 158P3D2 expression vectors.

[0604] The results demonstrate that CTL lines obtained from animalsprimed with peptide epitope recognize endogenously synthesized 158P3D2antigen. The choice of transgenic mouse model to be used for such ananalysis depends upon the epitope(s) that are being evaluated. Inaddition to HLA-A*0201/K^(b) transgenic mice, several other transgenicmouse models including mice with human A11, which may also be used toevaluate A3 epitopes, and B7 alleles have been characterized and others(e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1and HLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

[0605] This example illustrates the induction of CTLs and HTLs intransgenic mice, by use of a 158P3D2-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 158P3D2-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

[0606] Immunization procedures: Immunization of transgenic mice isperformed as described (Alexander et al., J. Immunol. 159:47534761,1997). For example, A2/1 K^(b) mice, which are transgenic for the humanHLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201motif- or HLA-A2 supermotif-bearing epitopes, and are primedsubcutaneously (base of the tail) with a 0.1 ml of peptide in IncompleteFreund's Adjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

[0607] Cell lines: Target cells for peptide-specific cytotoxicity assaysare Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene(e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)

[0608] In vitro CTL activation: One week after priming, spleen cells(30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic,irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask)in 10 ml of culture medium/T25 flask. After six days, effector cells areharvested and assayed for cytotoxic activity.

[0609] Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×10⁶=18 LU.

[0610] The results are analyzed to assess the magnitude of the CTLresponses of animals injected with the immunogenic CTL/HTL conjugatevaccine preparation and are compared to the magnitude of the CTLresponse achieved using, for example, CTL epitopes as outlined above inExample 14. Analyses similar to this may be performed to confirm theimmunogenicity of peptide conjugates containing multiple CTL epitopesand/or multiple HTL epitopes. In accordance with these procedures, it isfound that a CTL response is induced, and concomitantly that an HTLresponse is induced upon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in an158P3D2-Specific Vaccine

[0611] This example illustrates a procedure for selecting peptideepitopes for vaccine compositions of the invention. The peptides in thecomposition can be in the form of a nucleic acid sequence, either singleor one or more sequences (i.e., minigene) that encodes peptide(s), orcan be single and/or polyepitopic peptides.

[0612] The following principles are utilized when selecting a pluralityof epitopes for inclusion in a vaccine composition. Each of thefollowing principles is balanced in order to make the selection.

[0613] Epitopes are selected which, upon administration, mimic immuneresponses that are correlated with 158P3D2 clearance. The number ofepitopes used depends on observations of patients who spontaneouslyclear 158P3D2. For example, if it has been observed that patients whospontaneously clear 158P3D2 generate an immune response to at leastthree (3) from 158P3D2 antigen, then three or four (3-4) epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

[0614] Epitopes are often selected that have a binding affinity of anIC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, anIC₅₀ of 1000 nM or less; or HLA Class I peptides with high bindingscores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.

[0615] In order to achieve broad coverage of the vaccine through out adiverse population, sufficient supermotif bearing peptides, or asufficient array of allele-specific motif bearing peptides, are selectedto give broad population coverage. In one embodiment, epitopes areselected to provide at least 80% population coverage. A Monte Carloanalysis, a statistical evaluation known in the art, can be employed toassess breadth, or redundancy, of population coverage.

[0616] When creating polyepitopic compositions, or a minigene thatencodes same, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 158P3D2, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

[0617] A vaccine composition comprised of selected peptides, whenadministered, is safe, efficacious, and elicits an immune responsesimilar in magnitude to an immune response that controls or clears cellsthat bear or overexpress 158P3D2.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

[0618] This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

[0619] A minigene expression plasmid typically includes multiple CTL andHTL peptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 158P3D2, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 158P3D2 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

[0620] Such a construct may additionally include sequences that directthe HTL epitopes to the endoplasmic reticulum. For example, the Iiprotein may be fused to one or more HTL epitopes as described in theart, wherein the CLIP sequence of the Ii protein is removed and replacedwith an HLA class II epitope sequence so that HLA class II epitope isdirected to the endoplasmic reticulum, where the epitope binds to an HLAclass II molecules.

[0621] This example illustrates the methods to be used for constructionof a minigene-bearing expression plasmid. Other expression vectors thatmay be used for minigene compositions are available and known to thoseof skill in the art.

[0622] The minigene DNA plasmid of this example contains a consensusKozak sequence and a consensus murine kappa Ig-light chain signalsequence followed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

[0623] Overlapping oligonucleotides that can, for example, average about70 nucleotides in length with 15 nucleotide overlaps, are synthesizedand HPLC-purified. The oligonucleotides encode the selected peptideepitopes as well as appropriate linker nucleotides, Kozak sequence, andsignal sequence. The final multiepitope minigene is assembled byextending the overlapping oligonucleotides in three sets of reactionsusing PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30cycles are performed using the following conditions: 95° C. for 15 sec,annealing temperature (5° below the lowest calculated Tm of each primerpair) for 30 sec, and 72° C. for 1 min.

[0624] For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to Which it InducesImmunogenicity

[0625] The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

[0626] Alternatively, immunogenicity is confirmed through in vivoinjections into mice and subsequent in vitro assessment of CTL and HTLactivity, which are analyzed using cytotoxicity and proliferationassays, respectively, as detailed e.g., in Alexander et al., Immunity1:751-761, 1994.

[0627] For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

[0628] Splenocytes from immunized animals are stimulated twice with eachof the respective compositions (peptide epitopes encoded in the minigeneor the polyepitopic peptide), then assayed for peptide-specificcytotoxic activity in a ⁵¹Cr release assay. The results indicate themagnitude of the CTL response directed against the A2-restrictedepitope, thus indicating the in vivo immunogenicity of the minigenevaccine and polyepitopic vaccine.

[0629] It is, therefore, found that the minigene elicits immuneresponses directed toward the HLA-A2 supermotif peptide epitopes as doesthe polyepitopic peptide vaccine. A similar analysis is also performedusing other HLA-A3 and HLA-B7 transgenic mouse models to assess CTLinduction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby itis also found that the minigene elicits appropriate immune responsesdirected toward the provided epitopes.

[0630] To confirm the capacity of a class II epitope-encoding minigeneto induce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-Ab^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4⁺ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

[0631] DNA minigenes, constructed as described in the previous Example,can also be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

[0632] For example, the efficacy of the DNA minigene used in a primeboost protocol is initially evaluated in transgenic mice. In thisexample, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of aDNA minigene encoding the immunogenic peptides including at least oneHLA-A2 supermotif-bearing peptide. After an incubation period (rangingfrom 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of arecombinant vaccinia virus expressing the same sequence encoded by theDNA minigene. Control mice are immunized with 100 μg of DNA orrecombinant vaccinia without the minigene sequence, or with DNA encodingthe minigene, but without the vaccinia boost. After an additionalincubation period of two weeks, splenocytes from the mice areimmediately assayed for peptide-specific activity in an ELISPOT assay.Additionally, splenocytes are stimulated in vitro with the A2-restrictedpeptide epitopes encoded in the minigene and recombinant vaccinia, thenassayed for peptide-specific activity in an alpha, beta and/or gamma IFNELISA.

[0633] It is found that the minigene utilized in a prime-boost protocolelicits greater immune responses toward the HLA-A2 supermotif peptidesthan with DNA alone. Such an analysis can also be performed usingHLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction byHLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boostprotocols in humans is described below in Example 31.

Example 24 Peptide Compositions for Prophylactic Uses

[0634] Vaccine compositions of the present invention can be used toprevent 158P3D2 expression in persons who are at risk for tumors thatbear this antigen. For example, a polyepitopic peptide epitopecomposition (or a nucleic acid comprising the same) containing multipleCTL and HTL epitopes such as those selected in the above Examples, whichare also selected to target greater than 80% of the population, isadministered to individuals at risk for a 158P3D2-associated tumor.

[0635] For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against158P3D2-associated disease.

[0636] Alternatively, a composition typically comprising transfectingagents is used for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 158P3D2Sequences

[0637] A native 158P3D2 polyprotein sequence is analyzed, preferablyusing computer algorithms defined for each class I and/or class IIsupermotif or motif, to identify “relatively short” regions of thepolyprotein that comprise multiple epitopes. The “relatively short”regions are preferably less in length than an entire native antigen.This relatively short sequence that contains multiple distinct oroverlapping, “nested” epitopes is selected; it can be used to generate aminigene construct. The construct is engineered to express the peptide,which corresponds to the native protein sequence. The “relatively short”peptide is generally less than 250 amino acids in length, often lessthan 100 amino acids in length, preferably less than 75 amino acids inlength, and more preferably less than 50 amino acids in length. Theprotein sequence of the vaccine composition is selected because it hasmaximal number of epitopes contained within the sequence, i.e., it has ahigh concentration of epitopes. As noted herein, epitope motifs may benested or overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9-mer epitopes and one 10-merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

[0638] The vaccine composition will include, for example, multiple CTLepitopes from 158P3D2 antigen and at least one HTL epitope. Thispolyepitopic native sequence is administered either as a peptide or as anucleic acid sequence which encodes the peptide. Alternatively, ananalog can be made of this native sequence, whereby one or more of theepitopes comprise substitutions that alter the cross-reactivity and/orbinding affinity properties of the polyepitopic peptide.

[0639] The embodiment of this example provides for the possibility thatan as yet undiscovered aspect of immune system processing will apply tothe native nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 158P3D2, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

[0640] Related to this embodiment, computer programs are available inthe art which can be used to identify in a target sequence, the greatestnumber of epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

[0641] The 158P3D2 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 158P3D2 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 158P3D2 as well astumor-associated antigens that are often expressed with a target cancerassociated with 158P3D2 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

[0642] Peptides of the invention may be used to analyze an immuneresponse for the presence of specific antibodies, CTL or HTL directed to158P3D2. Such an analysis can be performed in a manner described by Ogget al., Science 279:2103-2106, 1998. In this Example, peptides inaccordance with the invention are used as a reagent for diagnostic orprognostic purposes, not as an immunogen.

[0643] In this example highly sensitive human leukocyte antigentetrameric complexes (“tetramers”) are used for a cross-sectionalanalysis of, for example, 158P3D2 HLA-A*0201-specific CTL frequenciesfrom HLA A*0201-positive individuals at different stages of disease orfollowing immunization comprising an 158P3D2 peptide containing anA*0201 motif. Tetrameric complexes are synthesized as described (Museyet al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavychain (A*0201 in this example) and β2-microglobulin are synthesized bymeans of a prokaryotic expression system. The heavy chain is modified bydeletion of the transmembrane-cytosolic tail and COOH-terminal additionof a sequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

[0644] For the analysis of patient blood samples, approximately onemillion PBMCs are centrifuged at 300 g for 5 minutes and resuspended in50 μl of cold phosphate-buffered saline. Tri-color analysis is performedwith the tetramer-phycoerythrin, along with anti-CD8-Tricolor, andanti-CD38. The PBMCs are incubated with tetramer and antibodies on icefor 30 to 60 min and then washed twice before formaldehyde fixation.Gates are applied to contain >99.98% of control samples. Controls forthe tetramers include both A*0201-negative individuals andA*0201-positive non-diseased donors. The percentage of cells stainedwith the tetramer is then determined by flow cytometry. The resultsindicate the number of cells in the PBMC sample that containepitope-restricted CTLs, thereby readily indicating the extent of immuneresponse to the 158P3D2 epitope, and thus the status of exposure to158P3D2, or exposure to a vaccine that elicits a protective ortherapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

[0645] The peptide epitopes of the invention are used as reagents toevaluate T cell responses, such as acute or recall responses, inpatients. Such an analysis may be performed on patients who haverecovered from 158P3D2-associated disease or who have been vaccinatedwith an 158P3D2 vaccine.

[0646] For example, the class I restricted CTL response of persons whohave been vaccinated may be analyzed. The vaccine may be any 158P3D2vaccine. PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

[0647] PBMC from vaccinated individuals are separated onFicoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended inRPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM),penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM)containing 10% heat-inactivated human AB serum (complete RPMI) andplated using microculture formats. A synthetic peptide comprising anepitope of the invention is added at 10 μg/ml to each well and HBV core128-140 epitope is added at 1 μg/ml to each well as a source of T cellhelp during the first week of stimulation.

[0648] In the microculture format, 4×10⁵ PBMC are stimulated withpeptide in 8 replicate cultures in 96-well round bottom plate in 100l/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and20 U/ml final concentration of rIL-2 are added to each well. On day 7the cultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

[0649] Target cell lines are autologous and allogeneic EBV-transformedB-LCL that are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

[0650] Cytotoxicity assays are performed in the following manner. Targetcells consist of either allogeneic HLA-matched or autologousEBV-transformed B lymphoblastoid cell line that are incubated overnightwith the synthetic peptide epitope of the invention at 10 μM, andlabeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.)for 1 hour after which they are washed four times with HBSS.

[0651] Cytolytic activity is determined in a standard 4-h, split well⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

[0652] The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 158P3D2 or an 158P3D2 vaccine.

[0653] Similarly, Class II restricted HTL responses may also beanalyzed. Purified PBMC are cultured in a 96-well flat bottom plate at adensity of 1.5×10⁵ cells/well and are stimulated with 10 μg/milsynthetic peptide of the invention, whole 158P3D2 antigen, or PHA. Cellsare routinely plated in replicates of 4-6 wells for each condition.After seven days of culture, the medium is removed and replaced withfresh medium containing 10U/ml IL-2. Two days later, 1 μCi ³H-thymidineis added to each well and incubation is continued for an additional 18hours. Cellular DNA is then harvested on glass fiber mats and analyzedfor ³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

[0654] A human clinical trial for an immunogenic composition comprisingCTL and HTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

[0655] A total of about 27 individuals are enrolled and divided into 3groups:

[0656] Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

[0657] Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

[0658] Group III: 3 subjects are injected with placebo and 6 subjectsare injected with 500 μg of peptide composition.

[0659] After 4 weeks following the first injection, all subjects receivea booster inoculation at the same dosage.

[0660] The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

[0661] Safety: The incidence of adverse events is monitored in theplacebo and drug treatment group and assessed in terms of degree andreversibility.

[0662] Evaluation of Vaccine Efficacy: For evaluation of vaccineefficacy, subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

[0663] The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 158P3D2

[0664] Phase II trials are performed to study the effect ofadministering the CTL-HTL peptide compositions to patients having cancerthat expresses 158P3D2. The main objectives of the trial are todetermine an effective dose and regimen for inducing CTLs in cancerpatients that express 158P3D2, to establish the safety of inducing a CTLand HTL response in these patients, and to see to what extent activationof CTLs improves the clinical picture of these patients, as manifested,e.g., by the reduction and/or shrinking of lesions. Such a study isdesigned, for example, as follows:

[0665] The studies are performed in multiple centers. The trial designis an open-label, uncontrolled, dose escalation protocol wherein thepeptide composition is administered as a single dose followed six weekslater by a single booster shot of the same dose. The dosages are 50, 500and 5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

[0666] There are three patient groupings. The first group is injectedwith 50 micrograms of the peptide composition and the second and thirdgroups with 500 and 5,000 micrograms of peptide composition,respectively. The patients within each group range in age from 21-65 andrepresent diverse ethnic backgrounds. All of them have a tumor thatexpresses 158P3D2.

[0667] Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 158P3D2-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

[0668] A prime boost protocol similar in its underlying principle tothat used to confirm the efficacy of a DNA vaccine in transgenic mice,such as described above in Example 23, can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

[0669] For example, the initial immunization may be performed using anexpression vector, such as that constructed in Example 22 in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

[0670] Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against158P3D2 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

[0671] Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 158P3D2 protein from which the epitopes in thevaccine are derived.

[0672] For example, a cocktail of epitope-comprising peptides isadministered ex vivo to PBMC, or isolated DC therefrom. A pharmaceuticalto facilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

[0673] As appreciated clinically, and readily determined by one of skillbased on clinical outcomes, the number of DC reinfused into the patientcan vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

[0674] In some embodiments, peptide-loaded PBMC are injected intopatients without purification of the DC. For example, PBMC generatedafter treatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

[0675] Ex vivo Activation of CTL/HTL Responses

[0676] Alternatively, ex vivo CTL or HTL responses to 158P3D2 antigenscan be induced by incubating, in tissue culture, the patient's, orgenetically compatible, CTL or HTL precursor cells together with asource of APC, such as DC, and immunogenic peptides. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused into the patient, where they will destroy (CTL) orfacilitate destruction (HTL) of their specific target cells, i.e., tumorcells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

[0677] Another method of identifying and confirming motif-bearingpeptides is to elute them from cells bearing defined MHC molecules. Forexample, EBV transformed B cell lines used for tissue typing have beenextensively characterized to determine which HLA molecules they express.In certain cases these cells express only a single type of HLA molecule.These cells can be transfected with nucleic acids that express theantigen of interest, e.g. 158P3D2. Peptides produced by endogenousantigen processing of peptides produced as a result of transfection willthen bind to HLA molecules within the cell and be transported anddisplayed on the cell's surface. Peptides are then eluted from the HLAmolecules by exposure to mild acid conditions and their amino acidsequence determined, e.g., by mass spectral analysis (e.g., Kubo et al.,J. Immunol. 152:3913, 1994). Because the majority of peptides that binda particular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

[0678] Alternatively, cell lines that do not express endogenous HLAmolecules can be transfected with an expression construct encoding asingle HLA allele. These cells can then be used as described, i.e., theycan then be transfected with nucleic acids that encode 158P3D2 toisolate peptides corresponding to 158P3D2 that have been presented onthe cell surface. Peptides obtained from such an analysis will bearmotif(s) that correspond to binding to the single HLA allele that isexpressed in the cell.

[0679] As appreciated by one in the art, one can perform a similaranalysis on a cell bearing more than one HLA allele and subsequentlydetermine peptides specific for each HLA allele expressed. Moreover, oneof skill would also recognize that means other than transfection, suchas loading with a protein antigen, can be used to provide a source ofantigen to the cell.

Example 34 Complementary Polynucleotides

[0680] Sequences complementary to the 158P3D2-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 158P3D2. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 158P3D2. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 158P3D2-encodingtranscript.

Example 35 Purification of Naturally-Occurring or Recombinant 158P3D2Using 158P3D2 Specific Antibodies

[0681] Naturally occurring or recombinant 158P3D2 is substantiallypurified by immunoaffinity chromatography using antibodies specific for158P3D2. An immunoaffinity column is constructed by covalently couplinganti-158P3D2 antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

[0682] Media containing 158P3D2 are passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of 158P3D2 (e.g., high ionic strength buffers inthe presence of detergent). The column is eluted under conditions thatdisrupt antibody/158P3D2 binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules Which Interact with 158P3D2

[0683] 158P3D2, or biologically active fragments thereof, are labeledwith 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973)Biochem. J. 133:529.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled 158P3D2,washed, and any wells with labeled 158P3D2 complex are assayed. Dataobtained using different concentrations of 158P3D2 are used to calculatevalues for the number, affinity, and association of 158P3D2 with thecandidate molecules.

Example 37 In vivo Assay for 158P3D2 Tumor Growth Promotion

[0684] The effect of the 158P3D2 protein on tumor cell growth isevaluated in vivo by gene overexpression in tumor-bearing mice. Forexample, SCID mice are injected subcutaneously on each flank with 1×10⁶of either NIH-3T3 cells, bladder cancer lines (UM-UC3, J82 or SCABER)and kidney cancer cells (CaKil, 769-P) containing tkNeo empty vector or158P3D2. At least two strategies may be used: (1) Constitutive 158P3D2expression under regulation of a promoter such as a constitutivepromoter obtained from the genomes of viruses such as polyoma virus,fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, provided such promoters arecompatible with the host cell systems, and (2) Regulated expressionunder control of an inducible vector system, such as ecdysone, tet,etc., provided such promoters are compatible with the host cell systems.Tumor volume is then monitored at the appearance of palpable tumors andfollowed over time to determine if 158P3D2-expressing cells grow at afaster rate and whether tumors produced by 158P3D2-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs).

[0685] Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 158P3D2 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow.

[0686] The assay is also useful to determine the 158P3D2 inhibitoryeffect of candidate therapeutic compositions, such as for example,158P3D2 intrabodies, 158P3D2 antisense molecules and ribozymes.

Example 38 158P3D2 Monoclonal Antibody-Mediated Inhibition of BladderTumors in vivo

[0687] The significant expression of 158P3D2 in cancer tissues, itsrestrictive expression in normal tissues together with its expected cellsurface expression makes 158P3D2 an excellent target for antibodytherapy. Similarly, 158P3D2 is a target for T cell-based immunotherapy.Thus, the therapeutic efficacy of anti-158P3D2 mAbs in human bladdercancer xenograft mouse models is evaluated by using recombinant celllines UM-UC3-158P3D2 and J28-158P3D2. Similarly, anti-158P3D2 mAbs areevaluated in human kidney cancer xenograft models such as AGS-K3 andAGS-K6 and in recombinant kidney cell lines such as Caki-158P3D2.

[0688] Antibody efficacy on tumor growth and metastasis formation isstudied, e.g., in a mouse orthotopic bladder cancer xenograft models andmouse kidney xenograft models. The antibodies can be unconjugated, asdiscussed in this Example, or can be conjugated to a therapeuticmodality, as appreciated in the art. Anti-158P3D2 mAbs inhibit formationof both Caki-158P3D2 and UMUC3-158P3D2 tumor xenografts. Anti-158P3D2mAbs also retard the growth of established orthotopic tumors andprolonged survival of tumor-bearing mice. These results indicate theutility of anti-158P3D2 mAbs in the treatment of local and advancedstages of kidney and bladder cancer. (See, e.g., (Saffran, D., et al.,PNAS 10: 1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698).These results indicate the use of anti-158P3D2 mAbs in the treatment ofbladder and kidney cancer.

[0689] Administration of the anti-158P3D2 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 158P3D2 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-158P3D2 mAbs for the treatment of local and metastaticprostate cancer. This example demonstrates that unconjugated 158P3D2monoclonal antibodies are effective to inhibit the growth of humanbladder tumor xenografts and human kidney xenografts grown in SCID mice;accordingly a combination of such efficacious monoclonal antibodies isalso effective.

[0690] Tumor Inhibition Using Multiple Unconjugated 158P3D2 mAbsMaterials and Methods

[0691] 158P3D2 Monoclonal Antibodies:

[0692] Monoclonal antibodies are raised against 158P3D2 as described inExample 11. The antibodies are characterized by ELISA, Western blot,FACS, and immunoprecipitation for their capacity to bind 158P3D2.Epitope mapping data for the anti-158P3D2 mAbs, as determined by ELISAand Western analysis, recognize epitopes on the 158P3D2 protein.Immunohistochemical analysis of prostate cancer tissues and cells withthese antibodies is performed.

[0693] The monoclonal antibodies are purified from ascites or hybridomatissue culture supernatants by Protein-G Sepharose chromatography,dialyzed against PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofLAPC-9 prostate tumor xenografts.

[0694] Cancer Xenografts and Cell Lines

[0695] Human cancer xenograft models, such as bladder and kidney cancermodels, as well as ICR-severe combined immunodeficient (SCID) miceinjected with human cell lines expressing or lacking 158P3D2 are used toconfirm the role of 158P3D2 in tumor growth and progression. The bladderxenograft is passaged in 6- to 8-week-old male ICR-severe combinedimmunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant(Craft, N., et al., supra). The AGS-K3 and AGS-K6 kidney xenografts arealso passaged by subcutaneous implants in 6- to 8-week old SCID mice.Single-cell suspensions of tumor cells are prepared as described inCraft, et al. The bladder and kidney carcinoma cell lines UM-UC3,SCABER, J82, 769-P and CaKi (American Type Culture Collection) aremaintained in DMEM supplemented with L-glutamine and 10% FBS.

[0696] A UMUC3-158P3D2, J82-158P3D2, 769-P-158P3D2 and CaKi-158P3D2 cellpopulations are generated by retroviral gene transfer as described inHubert, R. S., et al., STEAP: a prostate-specific cell-surface antigenhighly expressed in human prostate tumors. Proc Natl Acad Sci USA, 1999.96(25): p. 14523-8. Anti-158P3D2 staining is detected by using anFITC-conjugated goat anti-mouse antibody (Southern BiotechnologyAssociates) followed by analysis on a Coulter Epics-XL f low cytometer.

[0697] Xenograft Mouse Models.

[0698] Subcutaneous (s.c.) tumors are generated by injection of 1×10 ⁶AGS-K3, AGS-K6, A UMUC3-158P3D2, SCABER-158P3D2, 769-P-158P3D2 andCaKi-158P3D2 cells mixed at a 1:1 dilution with Matrigel (CollaborativeResearch) in the right flank of male SCID mice. To test antibodyefficacy on tumor formation, i.p. antibody injections are started on thesame day as tumor-cell injections. As a control, mice are injected witheither purified mouse IgG (ICN) or PBS; or a purified monoclonalantibody that recognizes an irrelevant antigen not expressed in humancells. Tumor sizes are determined by vernier caliper measurements, andthe tumor volume is calculated as length×width×height. Mice with s.c.tumors greater than 1.5 cm in diameter are sacrificed. PSA levels aredetermined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).Circulating levels of anti-158P3D2 mAbs are determined by a captureELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran,D., et al., PNAS 10:1073-1078 orwww.pnas.org/cgi/doi/10.1073/pnas.051624698)

[0699] Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For bladder orthotopic studies, an incision is madethrough the abdominal muscles to expose the bladder. Cells (5×10⁵) mixedwith Matrigel are injected into the bladder in a 10-μl volume. Forkidney orthopotic models, an incision is made through the abdominalmuscles to expose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigelare injected under the kidney capsule. The mice are segregated intogroups for the appropriate treatments, with anti-158P3D2 or control mAbsbeing injected i.p.

[0700] Anti-158P3D2 mAbs Inhibit Growth of 158P3D2-ExpressingXenograft-Cancer Tumors

[0701] The effect of anti-158P3D2 mAbs on tumor formation is tested byusing bladder and kidney orthotopic models. As compared with the s.c.tumor model, the orthotopic model, which requires injection of tumorcells directly in the mouse bladder or kidney, respectively, results ina local tumor growth, development of metastasis in distal sites,deterioration of mouse health, and subsequent death (Saffran, D., etal., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90;Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make theorthotopic model more representative of human disease progression andallowed us to follow the therapeutic effect of mAbs on clinicallyrelevant end points.

[0702] Accordingly, tumor cells are injected into the mouse bladder orkidney, and 2 days later, the mice are segregated into two groups andtreated with either: a) 200-500 μg, of anti-158P3D2 Ab, or b) PBS threetimes per week for two to five weeks.

[0703] A major advantage of the orthotopic cancer model is the abilityto study the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against BTA, a bladder specific antigen(Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p.14523-8) or anti-G250 antibody for kidney cancer models.

[0704] Mice bearing established orthotopic tumors are administered 1000μg injections of either anti-158P3D2 mAb or PBS over a 4-week period.Mice in both groups are allowed to establish a high tumor burden, toensure a high frequency of metastasis formation in mouse lungs. Micethen are killed and their bladder/kidney and lungs are analyzed for thepresence of tumor cells by IHC analysis.

[0705] These studies demonstrate a broad anti-tumor efficacy ofanti-158P3D2 antibodies on initiation and progression of bladder andkidney cancer in xenograft mouse models. Anti-158P3D2 antibodies inhibittumor formation of both androgen-dependent and androgen-independenttumors as well as retarding the growth of already established tumors andprolong the survival of treated mice. Moreover, anti-158P3D2 mAbsdemonstrate a dramatic inhibitory effect on the spread of local prostatetumor to distal sites, even in the presence of a large tumor burden.Thus, anti-158P3D2 mAbs are efficacious on major clinically relevant endpoints (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies inHumans

[0706] Anti-158P3D2 monoclonal antibodies are safely and effectivelyused for diagnostic, prophylactic, prognostic and/or therapeuticpurposes in humans. Western blot and immunohistochemical analysis ofcancer tissues and cancer xenografts with anti-158P3D2 mAb show strongextensive staining in carcinoma but significantly lower or undetectablelevels in normal tissues. Detection of 158P3D2 in carcinoma and inmetastatic disease demonstrates the usefulness of the mAb as adiagnostic and/or prognostic indicator. Anti-158P3D2 antibodies aretherefore used in diagnostic applications such as immunohistochemistryof kidney biopsy specimens to detect cancer from suspect patients.

[0707] As determined by flow cytometry, anti-158P3D2 mAb specificallybinds to carcinoma cells. Thus, anti-158P3D2 antibodies are used indiagnostic whole body imaging applications, such asradioimmunoscintigraphy and radioimmunotherapy, (see, e.g., PotamianosS., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection oflocalized and metastatic cancers that exhibit expression of 158P3D2.Shedding or release of an extracellular domain of 158P3D2 into theextracellular milieu, such as that seen for alkaline phosphodiesteraseB10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnosticdetection of 158P3D2 by anti-158P3D2 antibodies in serum and/or urinesamples from suspect patients.

[0708] Anti-158P3D2 antibodies that specifically bind 158P3D2 are usedin therapeutic applications for the treatment of cancers that express158P3D2. Anti-158P3D2 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-158P3D2 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., Example 38). Conjugated and unconjugated anti-158P3D2antibodies are used as a therapeutic modality in human clinical trialseither alone or in combination with other treatments as described infollowing Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-158P3D2 Antibodies in vivo

[0709] Antibodies are used in accordance with the present inventionwhich recognize an epitope on 158P3D2, and are used in the treatment ofcertain tumors such as those listedin Table I. Based upon a number offactors, including 158P3D2 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

[0710] I.) Adjunctive therapy: In adjunctive therapy, patients aretreated with anti-158P3D2 antibodies in combination with achemotherapeutic or antineoplastic agent and/or radiation therapy.Primary cancer targets, such as those listed in Table I, are treatedunder standard protocols by the addition anti-158P3D2 antibodies tostandard first and second line therapy. Protocol designs addresseffectiveness as assessed by reduction in tumor mass as well as theability to reduce usual doses of standard chemotherapy. These dosagereductions allow additional and/or prolonged therapy by reducingdose-related toxicity of the chemotherapeutic agent. Anti-158P3D2antibodies are utilized in several adjunctive clinical trials incombination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

[0711] II.) Monotherapy: In connection with the use of the anti-158P3D2antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

[0712] III.) Imaging Agent: Through binding a radionuclide (e.g., iodineor yttrium (I¹³¹, Y⁹⁰) to anti-158P3D2 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 158P3D2. In connection with theuse of the anti-158P3D2 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-158P3D2antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 158P3D2 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

[0713] Dose and Route of Administration

[0714] As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-158P3D2 antibodies can beadministered with doses in the range of 5 to 400 mg/m ², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-158P3D2 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-158P3D2 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-i 58P3D2 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

[0715] Three distinct delivery approaches are useful for delivery ofanti-158P3D2 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

[0716] Clinical Development Plan (CDP)

[0717] Overview: The CDP follows and develops treatments of anti-158P3D2antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-158P3D2antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 158P3D2 expression levels intheir tumors as determined by biopsy.

[0718] As with any protein or antibody infusion-based therapeutic,safety concerns are related primarily to (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 158P3D2.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-158P3D2 antibodies are found to be safe upon humanadministration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-158P3D2 Antibody and Chemotherapeutic Agent

[0719] A phase I human clinical trial is initiated to assess the safetyof six intravenous doses of a human anti-158P3D2 antibody in connectionwith the treatment of a solid tumor, e.g., a cancer of a tissue listedin Table I. In the study, the safety of single doses of anti-158P3D2antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin,adriamycin, taxol, or the like, is assessed. The trial design includesdelivery of six single doses of an anti-158P3D2 antibody with dosage ofantibody escalating from approximately about 25 mg/m² to about 275 mg/m²over the course of the treatment in accordance with the followingschedule: Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175225 275 mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + +(standard dose)

[0720] Patients are closely followed for one-week following eachadministration of antibody and chemotherapy. In particular, patients areassessed for the safety concerns mentioned above: (i) cytokine releasesyndrome, i.e., hypotension, fever, shaking, chills; (ii) thedevelopment of an immunogenic response to the material (i.e.,development of human antibodies by the patient to the human antibodytherapeutic, or HAHA response); and, (iii) toxicity to normal cells thatexpress 158P3D2. Standard tests and follow-up are utilized to monitoreach of these safety concerns. Patients are also assessed for clinicaloutcome, and particularly reduction in tumor mass as evidenced by MRI orother imaging.

[0721] The anti-158P3D2 antibodies are demonstrated to be safe andefficacious, Phase II trials confirm the efficacy and refine optimumdosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-158P3D2Antibody

[0722] Anti-158P3D2 antibodies are safe in connection with theabove-discussed adjunctive trial, a Phase II human clinical trialconfirms the efficacy and optimum dosing for monotherapy. Such trial isaccomplished, and entails the same safety and outcome analyses, to theabove-described adjunctive trial with the exception being that patientsdo not receive chemotherapy concurrently with the receipt of doses ofanti-158P3D2 antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-158P3D2Antibody

[0723] Once again, as the adjunctive therapy discussed above is safewithin the safety criteria discussed above, a human clinical trial isconducted concerning the use of anti-158P3D2 antibodies as a diagnosticimaging agent. The protocol is designed in a substantially similarmanner to those described in the art, such as in Divgi et al. J. Natl.Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safeand efficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 158P3D2 to Known Sequences

[0724] The 158P3D2 gene is identical to a previously cloned andsequenced gene, namely a novel protein similar to otoferlin anddysferlin, isoform 1 (gi 7671662), showing 100% identity to that protein(FIG. 4B). The 158P3D2 protein shows 65% homology and 45% identity tohuman otoferlin long isoform (gi 10119916), and 45% identity and 45%homology to the mouse otoferlin (gi 13994207) (FIGS. 4C and 4D,respectively). The 158P3D2 protein consists of 328 amino acids, withcalculated molecular weight of 38.4 kDa, and pI of 8.64. 158P3D2 is acell surface protein, with possible localization to the endoplasmicreticulum fraction. The 158P3D2 protein contains a single transmembranedomain at aa 145. Motif analysis revealed the presence of several knownmotifs, including a C2 domains located at the amino acids 122-144 of the158P3D2 protein, an aminoacyl-transfer RNA synthetases class II motif ataa 91-115. Pfam analysis suggests that 158P3D2 has a slight likelihoodof belonging to the chemokine receptor family (Table XXII).

[0725] C2 domains are Ca2+-binding motifs present in a variety ofproteins including phospholipases, protein kinases C and synaptotamins(Murakami M, et al Biochim Biophys Acta. 2000, 1488:159; Marqueze B etal, Biochimie. 2000, 82:409). They are about 116 amino-acid residueslong, and function in calcium-dependent phospholipid binding (StahelinRV, Cho W. Biochem J. 2001, 359:679). Since some C2-related domains arefound in proteins that do not bind calcium, C2 domains have beenassigned an additional function, namely inter-molecular association,such as binding to inositol-1,3,4,5-tetraphosphate (Mehrotra B et al,Biochemistry. 2000, 39:9679). C2 domains are also instrumental intargeting proteins to specific subcellular locations. In particular,recent studies have shown that the C2 domain of PLA mediates thetranslocation of PLA from the cytosol to the golgi in response tocalcium (Evans J H et al, J. Biol. Chem. 2001, 276:30150). In additionto affecting localization and protein association, C2 domain proteinshave been reported to regulate critical cellular functions, includingproliferation, a key component of tumoriogenesis (Koehler J A, Moran MF. Cell Growth Differ. 2001, 12:551).

[0726] Aminoacyl-tRNA synthetases are enzymes that activate amino acidsand transfer them to specific tRNA molecules as the first step inprotein biosynthesis (Fabrega C et al, Nature. 2001, 411:110). Ineukaryotes two aminoacyl-tRNA synthetases exist for each of the 20essential amino acid: a cytosolic form and a mitochondrial form. Theclass II synthetases are specific for alanine, asparagine, asparticacid, glycine, histidine, lysine, phenylalanine, proline, serine, andthreonine. Since aminoacyl transfer RNA synthetases regulate proteinsynthesis, it is clear that they also regulate cell proliferation andmaintain the accuracy of protein synthesis (Jakubowski H, Goldman E.Microbiol Rev. 1992, 56:412). This characteristic of aminoacyl transferRNA synthetases was used to develop reagents with anti-tumor effects invitro (Laske R et al, Arch Pharm. 1991, 324:153). The relevance ofaminoacyl transfer RNA synthetases to cell survival and growth wasdemonstrated in cells expressing mutant lysyl-tRNA synthetase. Mutationin lysyl-tRNA synthetases resulted in apoptosis of BHK21 cells(Fukushima et al, Genes Cells. 1996, 1:1087).

[0727] Based on the information above, 158P3D2 plays an important rolein several biological processes, including protein synthesis, cellgrowth, metabolism, and survival.

[0728] Several isoforms of 158P3D2 have been identified (FIG. 1). Whileboth variants var2a and var5a do not contain a transmembrane domain,var2a still maintains the C2 domain important for protein interaction,localization and calcium binding. Variant var2b still maintains thetransmembrane domain, but fails to exhibit a well-identified C2 domains.In addition, two variants, var3 and var4 contain a point mutations atamino acid 103 and 102, respectively, relative to the 158P3D2 var1protein. These single amino acid changes do not significantly alter thepredicted localization or motifs associated with 15 8P3D2 var1.

[0729] Accordingly, when any of the 158P3D2 variants function asregulators of protein synthesis, cell growth, metabolism, and survival,158P3D2 is used for therapeutic, diagnostic, prognostic and/orpreventative purposes.

Example 45 Identification and Confirmation of Potential SignalTransduction Pathways

[0730] Many mammalian proteins have been reported to interact withsignaling molecules and to participate in regulating signaling pathways(J. Neurochem. 2001; 76:217-223). In particular, C2-domain containingproteins have been reported to associate with signaling molecules andregulate signaling pathways including mitogenic cascades (Chow A et al,FEBS Lett. 2000;469:88; Walker E H et al, Nature. 1999,402:313). Usingimmunoprecipitation and Western blotting techniques, proteins areidentified that associate with 158P3D2 and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 158P3D2, including phospholipid pathways such as P13K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as wellas mitogenic/survival cascades such as ERK, p38, etc (Cell GrowthDiffer. 2000,11:279; J. Biol. Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.).

[0731] To confirm that 158P3D2 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressingindividual genes. These transcriptional reporters containconsensus-binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways. Thereporters and examples of these associated transcription factors, signaltransduction pathways, and activation stimuli arc listed below.

[0732] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

[0733] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

[0734] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

[0735] 4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

[0736] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

[0737] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

[0738] Gene-mediated effects can be assayed in cells showing mRNAexpression. Luciferase reporter plasmids can be introduced bylipid-mediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cell extracts with luciferin substrate and luminescence ofthe reaction is monitored in a luminometer.

[0739] Signaling pathways activated by 158P3D2 are mapped and used forthe identification and validation of therapeutic targets. When 158P3D2is involved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 46 Involvement in Tumor Progression

[0740] Based on the reported effect of C2 domains and tRNA synthetaseson cell growth, survival, protein regulation and signaling, the 158P3D2gene can contribute to the growth of cancer cells. The role of 158P3D2in tumor growth is confirmed in a variety of primary and transfectedcell lines including, bladder and kidney cell lines, as well as NIH 3T3cells engineered to stably express 158P3D2. Parental cells lacking158P3D2 and cells expressing 158P3D2 are evaluated for cell growth usinga well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz MB. Prostate. 2000;44:61, Johnson D E, Ochieng J, Evans S L. AnticancerDrugs. 1996, 7:288).

[0741] To confirm the role of 158P3D2 in the transformation process, itseffect in colony forming assays is investigated. Parental NIH-3T3 cellslacking 158P3D2 are compared to NIH-3T3 cells expressing 158P3D2, usinga soft agar assay under stringent and more permissive conditions (SongZ. et al. Cancer Res. 2000;60:6730).

[0742] To confirm the role of 158P3D2 in invasion and metastasis ofcancer cells, a well-established assay is used, e.g., a Transwell InsertSystem assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Controlcells, including bladder and kidney cell lines lacking 158P3D2 arecompared to cells expressing 158P3D2. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of the Transwellinsert coated with a basement membrane analog. Invasion is determined byfluorescence of cells in the lower chamber relative to the fluorescenceof the entire cell population.

[0743] 158P3D2 can also play a role in cell cycle and apoptosis.Parental cells and cells expressing 158P3D2 are compared for differencesin cell cycle regulation using a well-established BrdU assay(Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells aregrown under both optimal (full serum) and limiting (low serum)conditions are labeled with BrdU and stained with anti-BrdU Ab andpropidium iodide. Cells are analyzed for entry into the G1, S, and G2Mphases of the cell cycle. Alternatively, the effect of stress onapoptosis is evaluated in control parental cells and cells expressing158P3D2, including normal and tumor prostate, colon and lung cells.Engineered and parental cells are treated with various chemotherapeuticagents, such as etoposide, flutamide, etc, and protein synthesisinhibitors, such as cycloheximide. Cells are stained with annexin V-FITCand cell death is measured by FACS analysis. The modulation of celldeath by 158P3D2 can play a critical role in regulating tumorprogression and tumor load.

[0744] When 158P3D2 plays a role in cell growth, transformation,invasion or apoptosis, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Angiogenesis

[0745] Angiogenesis or new capillary blood vessel formation is necessaryfor tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of 158P3D2 on cellularfunctions and protein expression, 158P3D2 plays a role in angiogenesis.In addition, recent studies have associated human tyrosyl- andtryptophanyl-tRNA synthetases to angiogenesis (Otani A et al, Proc NatlAcad Sci U S A. 2002, 99:178). Several assays have been developed tomeasure angiogenesis in vitro and in vivo, such as the tissue cultureassays endothelial cell tube formation and endothelial cellproliferation. Using these assays as well as in vitroneo-vascularization, the role of 158P3D2 in angiogenesis, enhancement orinhibition, is confirmed.

[0746] For example, endothelial cells engineered to express 158P3D2 areevaluated using tube formation and proliferation assays. The effect of158P3D2 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 158P3D2 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. 158P3D2affects angiogenesis, and it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes

Example 48 Regulation of Protein Synthesis

[0747] The presence of a tRNA synthetase motif indicates that 158P3D2regulates protein synthesis. Regulation of protein synthesis isconfirmed, e.g., by studying gene expression in cells expressing orlacking 158P3D2. For this purpose, cells are labeled with ³H-Leucine andevaluated for the incorporation of the isotope (Tsurusaki Y, YamaguchiM. Int J Mol Med. 2000, 6:295). For examples cells lacking or expressing158P3D2 are incubated with ³H-Leucine for 6 hours in the presence ofabsence of stimuli such as growth factors, serum, phorbol esters. Cellsare lysed and evaluated for ³H-Leucine incorporation using abeta-counter (cpm).

[0748] Thus, 158P3D2 regulates protein synthesis, it is used as a targetfor diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 49 Protein-Protein Association

[0749] C2 domain-containing proteins have been shown to mediateprotein-protein association (Murakami M, et al Biochim Biophys Acta.2000, 1488:159; Chow A et al, FEBS Lett. 2000;469:88). Usingimmunoprecipitation techniques as well as two yeast hybrid systems,proteins are identified that associate with 158P3D2. Immunoprecipitatesfrom cells expressing 158P3D2 and cells lacking 158P3D2 are compared forspecific protein-protein associations.

[0750] Studies are performed to confirm the extent of association of158P3D2 with effector molecules, such as signaling intermediates,nuclear proteins, transcription factors, kinases, phosophates, etc.Studies comparing 158P3D2 positive and 158P3D2 negative cells as well asstudies comparing unstimulated/resting cells and cells treated withepithelial cell activators, such as cytokines, growth factors, androgenand anti-integrin Ab reveal unique interactions.

[0751] In addition, protein-protein interactions are confirmed using twoyeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vectorcarrying a library of proteins fused to the activation domain of atranscription factor is introduced into yeast expressing a158P3D2-DNA-binding domain fusion protein and a reporter construct.Protein-protein interaction is detected by calorimetric reporteractivity. Specific association with effector molecules and transcriptionfactors directs one of skill to the mode of action of 158P3D2, and thusidentifies therapeutic, prognostic, preventative and/or diagnostictargets for cancer. This and similar assays are also used to identifyand screen for small molecules that interact with 158P3D2.

[0752] Thus it is found that 158P3D2 associates with proteins and smallmolecules. Accordingly, 158P3D2 and these proteins and small moleculesare used for diagnostic, prognostic, preventative and/or therapeuticpurposes.

[0753] Throughout this application, various website data content,publications, patent applications and patents are referenced. (Websitesare referenced by their Uniform Resource Locator, or URL, addresses onthe World Wide Web.) The disclosures of each of these references arehereby incorporated by reference herein in their entireties.

[0754] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLES

[0755] TABLE I Tissues that Express 158P3D2 When Malignant ProstateBladder Kidney Colon Ovary Lung Breast Pancreas

[0756] TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULLNAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

[0757] TABLE III Amino Acid Substitution Matrix Adapted from the GCGSoftware 9.0 BLOSUM62 amino acid substitution matrix (block substitutionmatrix). The higher the value, the more likely a substitution is foundin related, natural proteins. (See URLwww.ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S TV W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4−2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4−3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2−2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3−3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2−2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7−1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3−2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

[0758] TABLE IV HLA Class I/II Motifs/Supermotifs TABLE IV (A): HLAClass I Supermotifs/Motifs POSITION POSITION POSITION C Terminus 2(Primary 3 (Primary (Primary Anchor) Anchor) Anchor) SUPERMOTIFS A1 TILVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 PYILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QLIVMP FWYMIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YF WM FLIW A*3101 MVTALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMF WYAIVB*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIVLMFWY

[0759] Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered notif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table. TABLE IV(B) HLA Class II Supermotif 1 6 9W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

[0760] TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1°anchor 6 7 8 9 DR4 preferred FMYLIVW M T W I VSTCPALIM MH MH deleteriousR WDE DR1 preferred MFLIVWY C CH PAMQ CWD VMATSPLIC M D AVM deleteriousFD GDE DR7 preferred MFLIVWY M W A IVMSACTPL M N IV deleterious C G GRDG DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY DKRH preferred LIVMFAY DNQEST motif b preferred DR MFLIVWY VMSTACPLISupermotif

[0761] TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS POSITION: 1 23 4 5 6 7 8 C-terminus A1 $\frac{1{^\circ}\quad {Anchor}}{TILVMS}$

$\frac{1{^\circ}\quad {Anchor}}{FWY}$

A2 $\frac{1{^\circ}\quad {Anchor}}{LIVMATQ}$

$\frac{1{^\circ}\quad {Anchor}}{LIVMAT}$

A3 preferred $\frac{1{^\circ}\quad {Anchor}}{VSMATLI}$

YFW (4/5) YFW (3/5) YFW (4/5) P (4/5)$\frac{1{^\circ}\quad {Anchor}}{RK}$

deleterious DE (3/5); DE P (5/5) (4/5) A24$\frac{1{^\circ}\quad {Anchor}}{YFWIVLMT}$

$\frac{1{^\circ}\quad {Anchor}}{FIYWLM}$

B7 preferred FWY (5/5) LIVM (3/5) $\frac{1{^\circ}\quad {Anchor}}{P}$

FWY (4/5) FWY (3/5) $\frac{1{^\circ}\quad {Anchor}}{VILFMWYA}$

deleterious DE (3/5); DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5);A(3/5); QN(3/5) B27 $\frac{1{^\circ}\quad {Anchor}}{RHK}$

$\frac{1{^\circ}\quad {Anchor}}{FYLWMIVA}$

B44 $\frac{1{^\circ}\quad {Anchor}}{ED}$

$\frac{1{^\circ}\quad {Anchor}}{FWYLIMVA}$

B58 $\frac{1{^\circ}\quad {Anchor}}{ATS}$

$\frac{1{^\circ}\quad {Anchor}}{FWYLIVMA}$

B62 $\frac{1{^\circ}\quad {Anchor}}{QLIVMP}$

$\frac{1{^\circ}\quad {Anchor}}{FWYMIVLA}$

[0762] TABLE IV (E) HLA Class I Motifs 9 or POSITION: 1 2 3 4 5 6 7 8C-terminus C-terminus A1 9-mer preferred GFY W$\frac{1{^\circ}\quad {Anchor}}{STM}$

DEA YFW P DEQN YFW $\frac{1{^\circ}\quad {Anchor}}{Y}$

deleterious DE RHKLIVMP A G A A1 9-mer preferred GRHK ASTCLIVM$\frac{1{^\circ}\quad {Anchor}}{DEAS}$

GSTC ASTC LIVM DE $\frac{1{^\circ}\quad {Anchor}}{Y}$

deleterious A RHKDEPY DE PQN RHK PG GP FW A1 10-mer preferred YFW$\frac{1{^\circ}\quad {Anchor}}{STM}$

DEAQN A YFWQN PASTC GDE P $\frac{1{^\circ}\quad {Anchor}}{Y}$

deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A A1 10-mer preferred YFWSTCLIVM $\frac{1{^\circ}\quad {Anchor}}{DEAS}$

A YFW PG G YFW $\frac{1{^\circ}\quad {Anchor}}{Y}$

deletetious RHK RHKDEPY P G PRHK QN FW A2.1 9-mer preferred YFW$\frac{1{^\circ}\quad {Anchor}}{LMIVQAT}$

YFW STC YFW A P $\frac{1{^\circ}\quad {Anchor}}{VLIMAT}$

deleterious DEP DERKH RKH DERKH A2.1 10-mer preferred AYFW$\frac{1{^\circ}\quad {Anchor}}{LMIVQAT}$

LVIM G G FYWL VIM $\frac{1{^\circ}\quad {Anchor}}{VLIMAT}$

deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK$\frac{1{^\circ}\quad {Anchor}}{LMVISATFCGD}$

YFW PRHK YFW A YFW P $\frac{1{^\circ}\quad {Anchor}}{KYRHFA}$

deleterious DEP DE A11 preferred A$\frac{1{^\circ}\quad {Anchor}}{VTLMISAGNCDF}$

YFW YFW A YFW YFW P $\frac{1{^\circ}\quad {Anchor}}{KRYH}$

deleterious DEP A G A24 9-mer preferred YFWRHK$\frac{1{^\circ}\quad {Anchor}}{YFWM}$

STC YFW YFW $\frac{1{^\circ}\quad {Anchor}}{FLIW}$

deleterious DEG DE G QNP DERH G AQN K A24 10-mer preferred$\frac{1{^\circ}\quad {Anchor}}{YFWM}$

P YFWP P $\frac{1{^\circ}\quad {Anchor}}{FLIW}$

deleterious GDE QN RHK DE A QN DEA A3101 preferred RHK$\frac{1{^\circ}\quad {Anchor}}{MVTALIS}$

YFW P YFW YFW AP $\frac{1{^\circ}\quad {Anchor}}{RK}$

deleterious DEP DE ADE DE DE DE A3301 preferred$\frac{1{^\circ}\quad {Anchor}}{MVALFIST}$

YFW AYFW $\frac{1{^\circ}\quad {Anchor}}{RK}$

deleterious GP DE A6801 preferred YFWSTC$\frac{1{^\circ}\quad {Anchor}}{AVTMSLI}$

YFWLIV M YFW P $\frac{1{^\circ}\quad {Anchor}}{RK}$

deleterious GP DEG RHK A B0702 preferred RHKFW Y$\frac{1{^\circ}\quad {Anchor}}{P}$

RHK RHK RHK RHK PA $\frac{1{^\circ}\quad {Anchor}}{LMFWYAIV}$

deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV M$\frac{1{^\circ}\quad {Anchor}}{P}$

FWY FWY $\frac{1{^\circ}\quad {Anchor}}{LMFWYIVA}$

deleterious AGP G G B51 preferred LIVMFW Y$\frac{1{^\circ}\quad {Anchor}}{P}$

FWY STC FWY G FWY $\frac{1{^\circ}\quad {Anchor}}{LIVFWYAM}$

deleterious AGPDER HKSTC DE G DEQN GDE B5301 preferred LIVMFW Y$\frac{1{^\circ}\quad {Anchor}}{P}$

FWY STC FWY LIVMFWY FWY $\frac{1{^\circ}\quad {Anchor}}{IMFWYALV}$

deleterious AGPQN G RHKQN DE B5401 preferred FWY$\frac{1{^\circ}\quad {Anchor}}{P}$

FWYL IVM LIVM ALIVM FWYAP $\frac{1{^\circ}\quad {Anchor}}{ATIVLMFWY}$

deleterious GPQNDE GDES TC RHKDE DE QNDGE DE

[0763] TABLE V 158P3D2 A1, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 123456789 Score NO. Table V: 158P3D2 v.1 A1-9-mers 222 FTDMGGNVY62.500 47 TGEMSSDIY 11.250 219 DLEFTDMGG 4.500 110 ALEEAEFRQ 4.500 237EAEFELLTV 4.500 247 EAEKRPVGK 3.600 198 AQEAQAGKK 2.700 78 TGEGNFNWR2.250 259 QPEPLEKPS 2.250 113 EAEFRQPAV 1.800 140 SLELQLPDM 1.800 281KTFVFFIWR 1.250 303 LTVFLLLVF 1.250 145 LPDMVRGAR 1.250 312 YTIPGQISQ1.250 69 ETDVHFNSL 1.250 34 NTEDVVLDD 1.125 320 QVIFRPLHK 1.000 166GAGPRCNLF 1.000 304 TVFLLLVFY 1.000 39 VLDDENPLT 1.000 188 LKEAEDVER0.900 235 KVEAEFELL 0.900 190 EAEDVEREA 0.900 62 GLEHDKQET 0.900 51SSDIYVKSW 0.750 2 WIDIFPQDV 0.500 257 RKQPEPLEK 0.500 142 ELQLPDMVR0.500 283 FVFFIWRRY 0.500 121 VLVLQVWDY 0.500 156 ELCSVQLAR 0.500 154GPELCSVQL 0.450 97 EREVSVWRR 0.450 242 LLTVEEAEK 0.400 197 EAQEAQAGK0.400 243 LTVEEAEKR 0.250 90 RFDYLPTER 0.250 49 EMSSDIYVK 0.200 4DIFPQDVPA 0.200 11 PAPPPVDIK 0.200 123 VLQVWDYDR 0.200 53 DIYVKSWVK0.200 262 PLEKPSRPK 0.180 75 NSLTGEGNF 0.150 67 KQETDVHFN 0.135 126VWDYDRISA 0.125 293 RTLVLLLLV 0.125 81 GNFNWRFVF 0.125 277 VNPLKTFVF0.125 77 LTGEGNFNW 0.125 214 KGRPEDLEF 0.125 270 KTSFNWFVN 0.125 85WRFVFRFDY 0.125 40 LDDENPLTG 0.125 216 RPEDLEFTD 0.113 298 LLLVLLTVF0.100 200 EAQAGKKKR 0.100 170 RCNLFRCRR 0.100 109 FALEEAEFR 0.100 276FVNPLKTFV 0.100 244 TVEEAEKRP 0.090 25 SYELRVVIW 0.090 193 DVEREAQEA0.090 195 EREAQEAQA 0.090 132 ISANDFLGS 0.075 316 GQISQVIFR 0.075 105RSGPFALEE 0.075 10 VPAPPPVDI 0.050 71 DVHFNSLTG 0.050 300 LVLLTVFLL0.050 137 FLGSLELQL 0.050 232 LTGKVEAEF 0.050 294 TLVLLLLVL 0.050 301VLLTVFLLL 0.050 302 LLTVFLLLV 0.050 227 GNVYILTGK 0.050 297 LLLLVLLTV0.050 296 VLLLLVLLT 0.050 131 RISANDFLG 0.050 308 LLVFYTIPG 0.050 245VEEAEKRPV 0.045 143 LQLPDMVRG 0.030 24 ISYELRVVI 0.030 201 AQAGKKKRK0.030 50 MSSDIYVKS 0.030 116 FRQPAVLVL 0.025 46 LTGEMSSDI 0.025 191AEDVEREAQ 0.025 95 PTEREVSVW 0.022 59 WVKGLEHDK 0.020 179 LRGWWPVVK0.020 306 FLLLVFYTI 0.020 157 LCSVQLARN 0.020 230 YILTGKVEA 0.020 309LVFYTIPGQ 0.020 299 LLVLLTVFL 0.020 17 DIKPRQPIS 0.020 295 LVLLLLVLL0.020 158 CSVQLARNG 0.015 Table V: 158P3D2 v.2a A1-9mers 180 ETELTVAVF45.000 203 HIDLENRFY 25.000 101 FSEPQISRG 13.500 138 KADPYVVVS 10.000 46SLEEEFNHF 9.000 93 YPESEAVLF 4.500 6 DSDGVNLIS 3.750 205 DLENRFYSH 1.800167 FGEILELSI 1.125 24 EAEVKGTVS 0.900 194 GSDDLIGET 0.750 95 ESEAVLFSE0.675 57 WLNVFPLYR 0.500 35 KAVATLKIY 0.500 53 HFEDWLNVF 0.450 109GIPQNRPIK 0.400 153 DTKERYIPK 0.250 201 ETHIDLENR 0.250 1 MDDPGDSDG0.250 73 GGEEEGSGH 0.225 22 QGEAEVKGT 0.225 37 VATLKIYNR 0.200 30TVSPKKAVA 0.200 130 LAPADPNGK 0.200 129 NLAPADPNG 0.200 19 IQDQGEAEV0.150 78 GSGHLVGKF 0.150 175 ISLPAETEL 0.150 216 ANCGLASQY 0.125 134DPNGKADPY 0.125 77 EGSGHLVGK 0.100 59 NVFPLYRGQ 0.100 162 QLNPIFGEI0.100 143 VVVSAGRER 0.100 91 LIYPESEAV 0.100 178 PAETELTVA 0.090 170ILELSISLP 0.090 187 VFEHDLVGS 0.090 45 RSLEEEFNH 0.075 151 RQDTKERYI0.075 9 GVNLISMVG 0.050 56 DWLNVFPLY 0.050 36 AVATLKIYN 0.050 182ELTVAVFEH 0.050 132 PADPNGKAD 0.050 198 LIGETHIDL 0.050 169 EILELSISL0.050 192 LVGSDDLIG 0.050 186 AVFEHDLVG 0.050 79 SGHLVGKFK 0.050 74GEEEGSGHL 0.045 75 EEEGSGHLV 0.045 223 QYEVWVQQG 0.045 118 LLVRVYVVK0.040 88 GSFLIYPES 0.030 173 LSISLPAET 0.030 195 SDDLIGETH 0.025 113NRPIKLLVR 0.025 150 ERQDTKERY 0.025 108 RGIPQNRPI 0.025 29 GTVSPKKAV0.025 100 LFSEPQISR 0.025 4 PGDSDGVNL 0.025 48 EEEFNHFED 0.022 16VGEIQDQGE 0.022 199 IGETHIDLE 0.022 98 AVLFSEPQI 0.020 121 RVYVVKATN0.020 220 LASQYEVWV 0.020 26 EVKGTVSPK 0.020 117 KLLVRVYVV 0.020 27VKGTVSPKK 0.020 215 RANCGLASQ 0.020 106 ISRGIPQNR 0.015 221 ASQYEVWVQ0.015 211 YSHHRANCG 0.015 228 VQQGPQEPF 0.015 85 KFKGSFLIY 0.013 112QNRPIKLLV 0.013 177 LPAETELTV 0.013 110 IPQNRPIKL 0.013 11 NLISMVGEI0.010 144 VVSAGRERQ 0.010 90 FLIYPESEA 0.010 12 LISMVGEIQ 0.010 99VLFSEPQIS 0.010 15 MVGEIQDQG 0.010 81 HLVGKFKGS 0.010 82 LVGKFKGSF 0.010191 DLVGSDDLI 0.010 184 TVAVFEHDL 0.010 20 QDQGEAEVK 0.010 185 VAVFEHDLV0.010 176 SLPAETELT 0.010 219 GLASQYEVW 0.010 97 EAVLFSEPQ 0.010 154TKERYIPKQ 0.009 69 GQDGGGEEE 0.007 13 ISMVGEIQD 0.007 115 PIKLLVRVY0.005 Table V: 158P3D2 v.3 A1-9mers 3 EREVSVRRR 0.450 1 PTEREVSVR 0.2255 EVSVRRRSG 0.010 7 SVRRRSGPF 0.001 2 TEREVSVRR 0.001 4 REVSVRRRS 0.0019 RRRSGPFAL 0.000 6 VSVRRRSGP 0.000 8 VRRRSGPFA 0.000 Table V: 158P3D2v.4 A1-9mers 4 EREVSIWRR 0.450 2 PTEREVSIW 0.022 6 EVSIWRRSG 0.010 1LPTEREVSI 0.005 3 TEREVSIWR 0.003 7 VSIWRRSGP 0.002 8 SIWRRSGPF 0.001 5REVSIWRRS 0.001 9 IWRRSGPFA 0.000 Table V: 158P3D2 v.5a A1-9mers 16SLDPWSCSY 250.000 28 CVGPGAPSS 0.200 8 YTASLPMTS 0.125 32 GAPSSALCS0.050 43 AMGPGRGAI 0.050 14 MTSLDPWSC 0.025 27 WCVGPGAPS 0.020 36SALCSWPAM 0.020 49 GAICFAAAA 0.020 37 ALCSWPAMG 0.020 2 VLQVWDYTA 0.02039 CSWPAMGPG 0.015 15 TSLDPWSCS 0.015 22 CSYQTWCVG 0.015 20 WSCSYQTWC0.015 10 ASLPMTSLD 0.015 35 SSALCSWPA 0.015 45 GPGRGAICF 0.013 21SCSYQTWCV 0.010 1 LVLQVWDYT 0.010 40 SWPAMGPGR 0.010 9 TASLPMTSL 0.01011 SLPMTSLDP 0.005 31 PGAPSSALC 0.005 38 LCSWPAMGP 0.005 48 RGAICFAAA0.005 44 MGPGRGAIC 0.005 25 QTWCVGPGA 0.005 6 WDYTASLPM 0.003 41WPAMGPGRG 0.003 29 VGPGAPSSA 0.003 5 VWDYTASLP 0.003 30 GPGAPSSAL 0.00333 APSSALCSW 0.003 12 LPMTSLDPW 0.003 47 GRGAICFAA 0.003 4 QVWDYTASL0.002 24 YQTWCVGPG 0.002 3 LQVWDYTAS 0.002 7 DYTASLPMT 0.001 13PMTSLDPWS 0.001 42 PAMGPGRGA 0.001 17 LDPWSCSYQ 0.001 18 DPWSCSYQT 0.00134 PSSALCSWP 0.000 23 SYQTWCVGP 0.000 26 TWCVGPGAP 0.000 19 PWSCSYQTW0.000 46 PGRGAICFA 0.000

[0764] TABLE VI 158P3D2 v.1 A1-10mers SEQ. ID Pos 1234567890 Score NO.Table VI: 158P3D2 A1, 10mers (variants 1, 2a, 3, 4 and 5a) 259QPEPLEKPSR 45.000 276 FVNPLKTFVF 5.000 166 GAGPRCNLFR 5.000 235KVEAEFELLT 4.500 198 AQEAQAGKKK 2.700 39 VLDDENPLTG 2.500 303 LTVFLLLVFY2.500 17 DIKPRQPISY 2.500 222 FTDMGGNVYI 2.500 78 TGEGNFNWRF 2.250 113EAEFRQPAVL 1.800 46 LTGEMSSDIY 1.250 69 ETDVHFNSLT 1.250 47 TGEMSSDIYV1.125 140 SLELQLPDMV 0.900 219 DLEFTDMGGN 0.900 190 EAEDVEREAQ 0.900 244TVEEAEKRPV 0.900 51 SSDIYVKSWV 0.750 67 KQETDVHFNS 0.675 134 ANDFLGSLEL0.625 120 AVLVLQVWDY 0.500 302 LLTVFLLLVF 0.500 10 VPAPPPVDIK 0.500 95PTEREVSVWR 0.450 241 ELLTVEEAEK 0.400 312 YTIPGQISQV 0.250 281KTFVFFIWRR 0.250 145 LPDMVRGARG 0.250 77 LTGEGNFNWR 0.250 12 APPPVDIKPR0.250 154 GPELCSVQLA 0.225 216 RPEDLEFTDM 0.225 34 NTEDVVLDDE 0.225 25SYELRVVIWN 0.225 122 LVLQVWDYDR 0.200 231 ILTGKVEAEF 0.200 197EAQEAQAGKK 0.200 200 EAQAGKKKRK 0.200 100 VSVWRRSGPF 0.150 105RSGPFALEEA 0.150 319 SQVIFRPLHK 0.150 80 EGNFNWRFVF 0.125 293 RTLVLLLLVL0.125 297 LLLLVLLTVF 0.100 144 QLPDMVRGAR 0.100 242 LLTVEEAEKR 0.100 193DVEREAQEAQ 0.090 247 EAEKRPVGKG 0.090 62 GLEHDKQETD 0.090 245 VEEAEKRPVG0.090 110 ALEEAEFRQP 0.090 237 EAEFELLTVE 0.090 107 GPFALEEAEF 0.050 15PVDIKPRQPI 0.050 304 TVFLLLVFYT 0.050 2 WIDIFPQDVP 0.050 76 SLTGEGNFNW0.050 307 LLLVFYTIPG 0.050 300 LVLLTVFLLL 0.050 295 LVLLLLVLLT 0.050 301VLLTVFLLLV 0.050 299 LLVLLTVFLL 0.050 261 EPLEKPSRPK 0.050 277VNPLKTPVFF 0.050 109 FALEEAEFRQ 0.050 81 GNFNWRFVFR 0.050 296 VLLLLVLLTV0.050 314 IPGQISQVIF 0.050 226 GGNVYILTGK 0.050 131 RISANDFLGS 0.050 97EREVSVWRRS 0.045 239 EFELLTVEEA 0.045 111 LEEAEFRQPA 0.045 41 DDENPLTGEM0.045 195 EREAQEAQAG 0.045 178 RLRGWWPVVK 0.040 24 ISYELRVVIW 0.030 139GSLELQLPDM 0.030 318 ISQVIFRPLH 0.030 224 DMGGNVYILT 0.025 165NGAGPRCNLF 0.025 282 TFVFFIWRRY 0.025 280 LKTFVFFIWR 0.025 82 NFNWRFVFRF0.025 171 CNLFRCRRLR 0.025 126 VWDYDRISAN 0.025 128 DYDRISANDF 0.025 141LELQLPDMVR 0.025 35 TEDVVLDDEN 0.025 74 FNSLTGEGNF 0.025 221 EFTDMGGNVY0.025 294 TLVLLLLVLL 0.020 38 VVLDDENPLT 0.020 142 ELQLPDMVRG 0.020 53DIYVKSWVKG 0.020 246 EEAEKRPVGK 0.020 187 KLKEAEDVER 0.020 272SFNWFVNPLK 0.020 298 LLLVLLTVFL 0.020 Table VI: 158P3D2 v.2a A1-10mers101 FSEPQISRGI 13.500 138 KADPYVVVSA 10.000 170 ILELSISLPA 4.500 6DSDGVNLISM 3.750 203 HIDLENRFYS 2.500 129 NLAPADPNGK 2.000 19 IQDQGEAEVK1.500 199 IGETHIDLEN 1.125 93 YPESEAVLFS 1.125 108 RGIPQNRPIK 1.000 205DLENRFYSHH 0.900 194 GSDDLIGETH 0.750 215 RANCGLASQY 0.500 99 VLFSEPQISR0.500 180 ETELTVAVFE 0.450 117 KLLVRVYVVK 0.400 78 GSGHLVGKFK 0.300 201ETHIDLENRF 0.250 1 MDDPGDSDGV 0.250 73 GGEEEGSGHL 0.225 16 VGEIQDQGEA0.225 22 QGEAEVKGTV 0.225 167 FGEILELSIS 0.225 75 EEEGSGHLVG 0.225 36AVATLKIYNR 0.200 30 TVSPKKAVAT 0.200 91 LIYPESEAVL 0.200 178 PAETELTVAV0.180 24 EAEVKGTVSP 0.180 175 ISLPAETELT 0.150 45 RSLEEEFNHF 0.150 95ESEAVLFSEP 0.135 112 QNRPIKLLVR 0.125 54 FEDWLNVFPL 0.125 132 PADPNGKADP0.100 81 HLVGKFKGSF 0.100 162 QLNPIFGEIL 0.100 59 NVFPLYRGQG 0.100 142YVVVSAGRER 0.100 227 WVQQGPQEPF 0.100 46 SLEEEFNHFE 0.090 69 GQDGGGEEEG0.075 140 DPYVVVSAGR 0.050 176 SLPAETELTV 0.050 197 DLIGETHIDL 0.050 35KAVATLKIYN 0.050 29 GTVSPKKAVA 0.050 185 VAVFEHDLVG 0.050 191 DLVGSDDLIG0.050 109 GIPQNRPIKL 0.050 148 GRERQDTKER 0.045 74 GEEEGSGHLV 0.045 48EEEFNHFEDW 0.045 26 EVKGTVSPKK 0.040 221 ASQYEVWVQQ 0.030 34 KKAVATLKIY0.025 195 SDDLIGETHI 0.025 77 EGSGHLVGKF 0.025 56 DWLNVFPLYR 0.025 133ADPNGKADPY 0.025 202 THIDLENRFY 0.025 127 ATNLAPADPN 0.025 183LTVAVFEHDL 0.025 189 EHDLVGSDDL 0.025 47 LEEEFNHFED 0.022 76 EEGSGHLVGK0.020 186 AVFEHDLVGS 0.020 217 NCGLASQYEV 0.020 172 ELSISLPAET 0.020 97EAVLFSEPQI 0.020 158 YIPKQLNPIF 0.020 57 WLNVFPLYRG 0.020 146 SAGRERQDTK0.020 18 EIQDQGEAEV 0.020 219 GLASQYEVWV 0.020 151 RQDTKERYIP 0.015 13ISMVGEIQDQ 0.015 145 VSAGRERQDT 0.015 211 YSHHRANCGL 0.015 84 GKFKGSFLIY0.013 79 SGHLVGKFKG 0.013 114 RPIKLLVRVY 0.013 164 NPIFGEILEL 0.013 8DGVNLISMVG 0.013 103 EPQISRGIPQ 0.013 51 FNHFEDWLNV 0.013 4 PGDSDGVNLI0.013 179 AETELTVAVF 0.010 92 IYPESEAVLF 0.010 174 SISLPAETEL 0.010 184TVAVFEHDLV 0.010 90 FLIYPESEAV 0.010 11 NLISMVGEIQ 0.010 98 AVLFSEPQIS0.010 121 RVYVVKATNL 0.010 37 VATLKIYNRS 0.010 143 VVVSAGRERQ 0.010 220LASQYEVWVQ 0.010 130 LAPADPNGKA 0.010 105 QISRGIPQNR 0.010 Table VI:158P3D2 v.3 A1-10mers 2 PTEREVSVRR 0.450 4 EREVSVRRRS 0.045 1 LPTEREVSVR0.025 7 VSVRRRSGPF 0.015 6 EVSVRRRSGP 0.001 3 TEREVSVRRR 0.001 5REVSVRRRSG 0.001 8 SVRRRSGPFA 0.000 9 VRRRSGPFAL 0.000 10 RRRSGPFALE0.000 Table VI: 158P3D2 v.4 A1-10mers 3 PTEREVSIWR 1.125 8 VSIWRRSGPF0.150 5 EREVSIWRRS 0.045 1 YLPTEREVSI 0.020 2 LPTEREVSIW 0.003 7EVSIWRRSGP 0.001 4 TEREVSIWRR 0.001 6 REVSIWRRSG 0.001 9 SIWRRSGPFA0.000 10 IWRRSGPFAL 0.000 Table VI: 158P3D2 v.5a A1-10mers 17 SLDPWSCSYQ5.000 16 TSLDPWSCSY 0.750 40 CSWPAMGPGR 0.300 45 MGPGRGAICF 0.125 6VWDYTASLPM 0.125 29 CVGPGAPSSA 0.100 44 AMGPGRGAIC 0.100 11 ASLPMTSLDP0.075 36 SSALCSWPAM 0.030 15 MTSLDPWSCS 0.025 9 YTASLPMTSL 0.025 28WCVGPGAPSS 0.020 2 LVLQVWDYTA 0.020 37 SALCSWPAMG 0.020 21 WSCSYQTWCV0.015 32 PGAPSSALCS 0.013 1 VLVLQVWDYT 0.010 12 SLPMTSLDPW 0.010 39LCSWPAMGPG 0.010 3 VLQVWDYTAS 0.010 33 GAPSSALCSW 0.010 22 SCSYQTWCVG0.010 49 RGAICFAAAA 0.005 38 ALCSWPAMGP 0.005 13 LPMTSLDPWS 0.005 31GPGAPSSALC 0.005 23 CSYQTWCVGP 0.003 4 LQVWDYTASL 0.003 25 YQTWCVGPGA0.003 8 DYTASLPMTS 0.003 42 WPAMGPGRGA 0.003 30 VGPGAPSSAL 0.003 35PSSALCSWPA 0.002 18 LDPWSCSYQT 0.001 27 TWCVGPGAPS 0.001 48 GRGAICFAAA0.001 10 TASLPMTSLD 0.001 7 WDYTASLPMT 0.001 43 PAMGPGRGAI 0.001 24SYQTWCVGPG 0.001 41 SWPAMGPGRG 0.001 14 PMTSLDPWSC 0.001 46 GPGRGAICFA0.000 26 QTWCVGPGAP 0.000 19 DPWSCSYQTW 0.000 34 APSSALCSWP 0.000 47PGRGAICFAA 0.000 5 QVWDYTASLP 0.000 20 PWSCSYQTWC 0.000

[0765] TABLE VII 158P3D2 A2, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 123456789 Score NO. Table VII: 158P3D2 v.1 A2-9mers 302 LLTVFLLLV1033.404 297 LLLLVLLTV 1006.209 286 FIWRRYWRT 440.113 306 FLLLVFYTI337.376 301 VLLTVFLLL 255.302 299 LLVLLTVFL 199.738 300 LVLLTVFLL156.843 276 FVNPLKTFV 153.971 296 VLLLLVLLT 107.808 137 FLGSLELQL 98.2672 WIDIFPQDV 66.867 38 VVLDDENPL 48.205 48 GEMSSDIYV 27.521 31 VIWNTEDVV27.109 295 LVLLLLVLL 27.042 313 TIPGQISQV 21.996 39 VLDDENPLT 20.776 294TLVLLLLVL 20.145 230 YILTGKVEA 11.626 144 QLPDMVRGA 9.370 293 RTLVLLLLV8.221 30 VVIWNTEDV 5.069 141 LELQLPDMV 4.168 236 VEAEFELLT 3.838 178RLRGWWPVV 3.684 94 LPTEREVSV 3.165 180 RGWWPVVKL 2.662 228 NVYILTGKV2.532 305 VFLLLVFYT 2.388 279 PLKTFVFFI 2.240 121 VLVLQVWDY 2.185 240FELLTVEEA 1.853 133 SANDFLGSL 1.382 124 LQVWDYDRI 1.322 224 DMGGNVYIL1.091 118 QPAVLVLQV 1.044 46 LTGEMSSDI 1.010 83 FNWRFVFRF 0.941 27ELRVVIWNT 0.733 140 SLELQLPDM 0.731 234 GKVEAEFEL 0.706 55 YVKSWVKGL0.692 114 AEFRQPAVL 0.630 24 ISYELRVVI 0.623 52 SDIYVKSWV 0.531 62GLEHDKQET 0.477 177 RRLRGWWPV 0.456 22 QPISYELRV 0.454 298 LLLVLLTVF0.442 159 SVQLARNGA 0.435 76 SLTGEGNFN 0.410 235 KVEAEFELL 0.390 183WPVVKLKEA 0.343 269 PKTSFNWFV 0.333 26 YELRVVIWN 0.312 304 TVFLLLVFY0.305 186 VKLKEAEDV 0.298 223 TDMGGNVYI 0.295 307 LLLVFYTIP 0.219 4DIFPQDVPA 0.190 165 NGAGPRCNL 0.139 272 SFNWFVNPL 0.130 308 LLVFYTIPG0.127 225 MGGNVYILT 0.124 10 VPAPPPVDI 0.116 112 EEAEFRQPA 0.113 135NDFLGSLEL 0.110 143 LQLPDMVRG 0.109 281 KTFVFFIWR 0.106 171 CNLFRCRRL0.103 8 QDVPAPPPV 0.097 318 ISQVIFRPL 0.090 87 FVFRFDYLP 0.084 86RFVFRFDYL 0.076 93 YLPTEREVS 0.069 80 EGNFNWRFV 0.064 131 RISANDFLG0.059 290 RYWRTLVLL 0.057 314 IPGQISQVI 0.047 77 LTGEGNFNW 0.042 79GEGNFNWRF 0.041 23 PISYELRVV 0.040 70 TDVHFNSLT 0.039 109 FALEEAEFR0.039 283 FVFFIWRRY 0.038 122 LVLQVWDYD 0.038 106 SGPFALEEA 0.037 68QETDVHFNS 0.034 168 GPRCNLFRC 0.033 292 WRTLVLLLL 0.031 245 VEEAEKRPV0.029 319 SQVIFRPLH 0.029 231 ILTGKVEAE 0.029 317 QISQVIFRP 0.027 120AVLVLQVWD 0.027 215 GRPEDLEFT 0.026 242 LLTVEEAEK 0.025 123 VLQVWDYDR0.025 16 VDIKPRQPI 0.025 258 KQPEPLEKP 0.024 Table VII: 158P3D2 v.2aA2-9mers 117 KLLVRVYVV 849.359 91 LIYPESEAV 25.492 90 FLIYPESEA 22.853198 LIGETHIDL 20.473 158 YIPKQLNPI 15.177 220 LASQYEVWV 9.032 184TVAVFEHDL 7.103 179 AETELTVAV 5.545 19 IQDQGEAEV 4.795 176 SLPAETELT3.651 98 AVLFSEPQI 3.378 169 EILELSISL 3.342 116 IKLLVRVYV 3.342 177LPAETELTV 3.165 11 NLISMVGEI 3.119 162 QLNPIFGEI 2.577 123 YVVKATNLA2.000 218 CGLASQYEV 1.680 57 WLNVFPLYR 1.433 52 NHFEDWLNV 1.246 114RPIKLLVRV 1.044 29 GTVSPKKAV 0.966 175 ISLPAETEL 0.877 185 VAVFEHDLV0.805 23 GEAEVKGTV 0.721 171 LELSISLPA 0.608 165 PIFGEILEL 0.550 151RQDTKERYI 0.465 191 DLVGSDDLI 0.383 84 GKFKGSFLI 0.311 161 KQLNPIFGE0.261 55 EDWLNVFPL 0.246 137 GKADPYVVV 0.244 110 IPQNRPIKL 0.237 99VLFSEPQIS 0.192 163 LNPIFGEIL 0.181 30 TVSPKKAVA 0.178 39 TLKIYNRSL0.150 5 GDSDGVNLI 0.137 119 LVRVYVVKA 0.129 28 KGTVSPKKA 0.114 155KERYIPKQL 0.110 111 PQNRPIKLL 0.110 146 SAGRERQDT 0.104 204 IDLENRFYS0.085 173 LSISLPAET 0.083 31 VSPKKAVAT 0.083 8 DGVNLISMV 0.078 182ELTVAVFEH 0.075 129 NLAPADPNG 0.075 135 PNGKADPYV 0.055 34 KKAVATLKI0.051 83 VGKFKGSFL 0.046 45 RSLEEEFNH 0.043 102 SEPQISRGI 0.041 186AVFEHDLVG 0.041 46 SLEEEFNHF 0.037 36 AVATLKIYN 0.036 112 QNRPIKLLV0.035 222 SQYEVWVQQ 0.034 125 VKATNLAPA 0.027 14 SMVGEIQDQ 0.025 194GSDDLIGET 0.024 105 QISRGIPQN 0.024 41 KIYNRSLEE 0.023 219 GLASQYEVW0.022 15 MVGEIQDQG 0.022 121 RVYVVKATN 0.021 167 FGEILELSI 0.020 131APADPNGKA 0.017 51 FNHFEDWLN 0.017 139 ADPYVVVSA 0.016 7 SDGVNLISM 0.016118 LLVRVYVVK 0.016 212 SHHRANCGL 0.015 74 GEEEGSGHL 0.014 206 LENRFYSHH0.014 108 RGIPQNRPI 0.014 17 GEIQDQGEA 0.013 50 EFNHFEDWL 0.011 32SPKKAVATL 0.011 92 IYPESEAVL 0.008 61 FPLYRGQGG 0.008 22 QGEAEVKGT 0.007136 NGKADPYVV 0.007 75 EEEGSGHLV 0.006 228 VQQGPQEPF 0.006 227 WVQQGPQEP0.006 181 TELTVAVFE 0.006 38 ATLKIYNRS 0.006 82 LVGKFKGSF 0.005 122VYVVKATNL 0.005 81 HLVGKFKGS 0.005 86 FKGSFLIYP 0.005 192 LVGSDDLIG0.005 120 VRVYVVKAT 0.004 196 DDLIGETHI 0.004 170 ILELSISLP 0.004 2DDPGDSDGV 0.004 35 KAVATLKIY 0.003 Table VII: 158P3D2 v.3 A2-9mers 9RRRSGPFAL 0.001 8 VRRRSGPFA 0.000 4 REVSVRRRS 0.000 6 VSVRRRSGP 0.000 5EVSVRRRSG 0.000 2 TEREVSVRR 0.000 7 SVRRRSGPF 0.000 1 PTEREVSVR 0.000 3EREVSVRRR 0.000 Table VII: 158P3D2 v.4 A2-9mers 1 LPTEREVSI 0.475 8SIWRRSGPF 0.011 3 TEREVSIWR 0.000 5 REVSIWRRS 0.000 9 IWRRSGPFA 0.000 7VSIWRRSGP 0.000 6 EVSIWRRSG 0.000 2 PTEREVSIW 0.000 4 EREVSIWRR 0.000Table VII: 158P3D2 v.5a A2-9mers 4 QVWDYTASL 63.609 1 LVLQVWDYT 18.791 2VLQVWDYTA 8.446 21 SCSYQTWCV 3.405 43 AMGPGRGAI 0.980 14 MTSLDPWSC 0.88020 WSCSYQTWC 0.820 9 TASLPMTSL 0.682 25 QTWCVGPGA 0.573 36 SALCSWPAM0.434 49 GAICFAAAA 0.262 35 SSALCSWPA 0.243 30 GPGAPSSAL 0.139 6WDYTASLPM 0.102 37 ALCSWPAMG 0.075 48 RGAICFAAA 0.062 29 VGPGAPSSA 0.05518 DPWSCSYQT 0.030 16 SLDPWSCSY 0.030 44 MGPGRGAIC 0.023 3 LQVWDYTAS0.019 11 SLPMTSLDP 0.015 15 TSLDPWSCS 0.013 24 YQTWCVGPG 0.010 28CVGPGAPSS 0.007 13 PMTSLDPWS 0.007 8 YTASLPMTS 0.005 47 GRGAICFAA 0.00412 LPMTSLDPW 0.003 27 WCVGPGAPS 0.002 39 CSWPAMGPG 0.001 42 PAMGPGRGA0.001 33 APSSALCSW 0.001 22 CSYQTWCVG 0.001 32 GAPSSALCS 0.001 31PGAPSSALC 0.001 46 PGRGAICFA 0.001 45 GPGRGAICF 0.000 10 ASLPMTSLD 0.00041 WPAMGPGRG 0.000 17 LDPWSCSYQ 0.000 7 DYTASLPMT 0.000 38 LCSWPAMGP0.000 34 PSSALCSWP 0.000 23 SYQTWCVGP 0.000 40 SWPAMGPGR 0.000 5VWDYTASLP 0.000 19 PWSCSYQTW 0.000 26 TWCVGPGAP 0.000

[0766] TABLE VIII 158P3D2 A2, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 1234567890 Score NO. Table VIII: 158P3D2 v.1 A2-10mers 301VLLTVFLLLV 3823.593 296 VLLLLVLLTV 1006.209 298 LLLVLLTVFL 739.032 299LLVLLTVFLL 484.457 93 YLPTEREVSV 319.939 304 TVFLLLVFYT 177.011 278NPLKTFVFFI 70.254 294 TLVLLLLVLL 49.134 26 YELRVVIWNT 42.542 286FIWRRYWRTL 38.130 300 LVLLTVFLLL 22.339 236 VEAEFELLTV 21.680 101SVWRRSGPFA 19.844 31 VIWNTEDVVL 16.993 38 VVLDDENPLT 16.816 87FVFRFDYLPT 16.647 117 RQPAVLVLQV 16.219 125 QVWDYDRISA 14.793 123VLQVWDYDRI 13.036 312 YTIPGQISQV 10.220 295 LVLLLLVLLT 9.433 63LEHDKQETDV 9.426 21 RQPISYELRV 7.052 114 AEFRQPAVLV 5.004 271 TSFNWFVNPL4.510 68 QETDVHFNSL 3.236 29 RVVIWNTEDV 2.982 61 KGLEHDKQET 2.583 79GEGNFNWRFV 2.529 268 RPKTSFNWFV 2.491 140 SLELQLPDMV 2.181 30 VVIWNTEDVV2.078 273 FNWFVNPLKT 1.857 222 FTDMGGNVYI 1.466 143 LQLPDMVRGA 1.457 275WFVNPLKTFV 1.222 139 GSLELQLPDM 1.132 317 QISQVIFRPL 1.116 220LEFTDMGGNV 1.106 293 RTLVLLLLVL 1.035 51 SSDIYVKSWV 0.999 309 LVFYTIPGQI0.746 224 DMGGNVYILT 0.605 306 FLLLVFYTIP 0.593 313 TIPGQISQVI 0.588 153RGPELCSVQL 0.572 235 KVEAEFELLT 0.555 307 LLLVFYTIPG 0.469 297LLLLVLLTVF 0.442 167 AGPRCNLFRC 0.433 76 SLTGEGNFNW 0.432 120 AVLVLQVWDY0.416 112 EEAEFRQPAV 0.416 244 TVEEAEKRPV 0.319 91 FDYLPTEREV 0.284 189KEAEDVEREA 0.277 172 NLFRCRRLRG 0.276 132 ISANDFLGSL 0.269 285FFIWRRYWRT 0.268 85 WRFVFRFDYL 0.259 1 MWIDIFPQDV 0.256 148 MVRGARGPEL0.242 45 PLTGEMSSDI 0.230 39 VLDDENPLTG 0.208 185 VVKLKEAEDV 0.177 281KTFVFFIWRR 0.176 151 GARGPELCSV 0.169 47 TGEMSSDIYV 0.160 137 FLGSLELQLP0.158 37 DVVLDDENPL 0.140 164 RNGAGPRCNL 0.139 231 ILTGKVEAEF 0.127 283FVFFIWRRYW 0.122 302 LLTVFLLLVF 0.119 121 VLVLQVWDYD 0.116 234GKVEAEFELL 0.113 258 KQPEPLEKPS 0.108 223 TDMGGNVYIL 0.104 292WRTLVLLLLV 0.102 305 VFLLLVFYTI 0.087 22 QPISYELRVV 0.086 109 FALEEAEFRQ0.084 214 KGRPEDLEFT 0.080 276 FVNPLKTFVF 0.071 9 DVPAPPPVDI 0.068 7PQDVPAPPPV 0.062 227 GNVYILTGKV 0.059 308 LLVFYTIPGQ 0.058 290RYWRTLVLLL 0.057 134 ANDFLGSLEL 0.056 194 VEREAQEAQA 0.051 111LEEAEFRQPA 0.040 230 YILTGKVEAE 0.039 19 KPRQPISYEL 0.037 105 RSGPFALEEA0.037 158 CSVQLARNGA 0.032 233 TGKVEAEFEL 0.028 129 YDRISANDFL 0.028 170RCNLFRCRRL 0.028 177 RRLRGWWPVV 0.025 Table VIII: 158P3D2 v.2a A2-10mers219 GLASQYEVWV 382.536 90 FLIYPESEAV 156.770 176 SLPAETELTV 69.552 118LLVRVYVVKA 19.425 82 LVGKFKGSFL 17.477 162 QLNPIFGEIL 16.308 54FEDWLNVFPL 10.196 91 LIYPESEAVL 6.551 121 RVYVVKATNL 5.981 51 FNHFEDWLNV3.550 161 KQLNPIFGEI 3.383 184 TVAVFEHDLV 2.982 18 EIQDQGEAEV 2.941 174SISLPAETEL 2.937 109 GIPQNRPIKL 2.937 183 LTVAVFEHDL 1.917 197DLIGETHIDL 1.602 28 KGTVSPKKAV 1.589 49 EEFNHFEDWL 1.180 57 WLNVFPLYRG0.788 30 TVSPKKAVAT 0.652 211 YSHHRANCGL 0.641 116 IKLLVRVYVV 0.573 172ELSISLPAET 0.559 31 VSPKKAVATL 0.545 110 IPQNRPIKLL 0.545 170 ILELSISLPA0.541 21 DQGEAEVKGT 0.534 217 NCGLASQYEV 0.454 168 GEILELSISL 0.415 74GEEEGSGHLV 0.355 164 NPIFGEILEL 0.321 222 SQYEVWVQQG 0.228 186AVFEHDLVGS 0.228 138 KADPYVVVSA 0.222 7 SDGVNLISMV 0.222 38 ATLKIYNRSL0.220 177 LPAETELTVA 0.213 119 LVRVYVVKAT 0.194 134 DPNGKADPYV 0.187 111PQNRPIKLLV 0.155 175 ISLPAETELT 0.150 10 VNLISMVGEI 0.128 117 KLLVRVYVVK0.119 193 VGSDDLIGET 0.101 99 VLFSEPQISR 0.094 145 VSAGRERQDT 0.083 46SLEEEFNHFE 0.082 181 TELTVAVFEH 0.072 124 VVKATNLAPA 0.059 166IFGEILELSI 0.050 3 DPGDSDGVNL 0.043 115 PIKLLVRVYV 0.041 1 MDDPGDSDGV0.032 29 GTVSPKKAVA 0.028 14 SMVGEIQDQG 0.026 41 KIYNRSLEEE 0.026 83VGKFKGSFLI 0.024 113 NRPIKLLVRV 0.022 35 KAVATLKIYN 0.020 158 YIPKQLNPIF0.019 198 LIGETHIDLE 0.016 130 LAPADPNGKA 0.015 129 NLAPADPNGK 0.015 227WVQQGPQEPF 0.015 45 RSLEEEFNHF 0.014 89 SFLIYPESEA 0.013 27 VKGTVSPKKA0.012 209 RFYSHHRANC 0.011 97 EAVLFSEPQI 0.011 98 AVLFSEPQIS 0.010 136NGKADPYVVV 0.010 15 MVGEIQDQGE 0.009 123 YVVKATNLAP 0.006 195 SDDLIGETHI0.006 179 AETELTVAVF 0.006 188 FEHDLVGSDD 0.005 169 EILELSISLP 0.005 192LVGSDDLIGE 0.005 204 IDLENRFYSH 0.005 73 GGEEEGSGHL 0.005 203 HIDLENRFYS0.004 171 LELSISLPAE 0.004 135 PNGKADPYVV 0.004 101 FSEPQISRGI 0.004 22QGEAEVKGTV 0.004 12 LISMVGEIQD 0.003 157 RYIPKQLNPI 0.003 59 NVFPLYRGQG0.003 9 GVNLISMVGE 0.003 36 AVATLKIYNR 0.003 11 NLISMVGEIQ 0.003 79SGHLVGKFKG 0.003 37 VATLKIYNRS 0.003 87 KGSFLIYPES 0.003 220 LASQYEVWVQ0.002 23 GEAEVKGTVS 0.002 191 DLVGSDDLIG 0.002 6 DSDGVNLISM 0.002 105QISRGIPQNR 0.002 Table VIII: 158P3D2 v.3 A2-10mers 8 SVRRRSGPFA 0.182 9VRRRSGPFAL 0.002 1 LPTEREVSVR 0.001 5 REVSVRRRSG 0.000 7 VSVRRRSGPF0.000 6 EVSVRRRSGP 0.000 3 TEREVSVRRR 0.000 10 RRRSGPFALE 0.000 2PTEREVSVRR 0.000 4 EREVSVRRRS 0.000 Table VIII: 158P3D2 v.4 A2-10mers 1YLPTEREVSI 47.991 9 SIWRRSGPFA 31.184 2 LPTEREVSIW 0.003 10 IWRRSGPFAL0.002 4 TEREVSIWRR 0.002 6 REVSIWRRSG 0.000 8 VSIWRRSGPF 0.000 7EVSIWRRSGP 0.000 3 PTEREVSIWR 0.000 5 EREVSIWRRS 0.000 Table VIII:158P3D2 v.5a A2-10mers 1 VLVLQVWDYT 58.040 21 WSCSYQTWCV 15.664 4LQVWDYTASL 3.682 9 YTASLPMTSL 3.139 2 LVLQVWDYTA 2.734 25 YQTWCVGPGA2.317 44 AMGPGRGAIC 1.471 14 PMTSLDPWSC 0.592 29 CVGPGAPSSA 0.435 46GPGRGAICFA 0.410 7 WDYTASLPMT 0.350 30 VGPGAPSSAL 0.237 3 VLQVWDYTAS0.190 49 RGAICFAAAA 0.123 12 SLPMTSLDPW 0.084 36 SSALCSWPAM 0.055 5QVWDYTASLP 0.044 17 SLDPWSCSYQ 0.033 31 GPGAPSSALC 0.032 42 WPAMGPGRGA0.030 18 LDPWSCSYQT 0.018 13 LPMTSLDPWS 0.017 38 ALCSWPAMGP 0.015 16TSLDPWSCSY 0.007 35 PSSALCSWPA 0.005 37 SALCSWPAMG 0.004 15 MTSLDPWSCS0.003 33 GAPSSALCSW 0.002 28 WCVGPGAPSS 0.002 43 PAMGPGRGAI 0.002 48GRGAICFAAA 0.001 45 MGPGRGAICF 0.001 40 CSWPAMGPGR 0.001 34 APSSALCSWP0.001 6 VWDYTASLPM 0.000 19 DPWSCSYQTW 0.000 11 ASLPMTSLDP 0.000 22SCSYQTWCVG 0.000 47 PGRGAICFAA 0.000 23 CSYQTWCVGP 0.000 39 LCSWPAMGPG0.000 26 QTWCVGPGAP 0.000 10 TASLPMTSLD 0.000 20 PWSCSYQTWC 0.000 32PGAPSSALCS 0.000 27 TWCVGPGAPS 0.000 24 SYQTWCVGPG 0.000 41 SWPAMGPGRG0.000 8 DYTASLPMTS 0.000

[0767] TABLE IX 158P3D2 A3, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 123456789 Score NO. Table IX: 158P3D2 v.1 A3-9-mers 281 KTFVFFIWR54.000 121 VLVLQVWDY 54.000 123 VLQVWDYDR 36.000 49 EMSSDIYVK 27.000 242LLTVEEAEK 20.000 306 FLLLVFYTI 12.150 53 DIYVKSWVK 9.000 301 VLLTVFLLL8.100 320 QVIFRPLHK 6.000 298 LLLVLLTVF 4.500 142 ELQLPDMVR 3.600 156ELCSVQLAR 3.600 316 GQISQVIFR 3.240 59 WVKGLEHDK 3.000 304 TVFLLLVFY3.000 294 TLVLLLLVL 2.700 224 DMGGNVYIL 2.430 172 NLFRCRRLR 2.000 302LLTVFLLLV 1.800 279 PLKTFVFFI 1.620 297 LLLLVLLTV 1.350 137 FLGSLELQL1.200 181 GWWPVVKLK 1.013 299 LLVLLTVFL 0.900 296 VLLLLVLLT 0.900 178RLRGWWPVV 0.900 300 LVLLTVFLL 0.810 81 GNFNWRFVF 0.540 235 KVEAEFELL0.540 83 FNWRFVFRF 0.540 303 LTVFLLLVF 0.450 243 LTVEEAEKR 0.450 201AQAGKKKRK 0.450 227 GNVYILTGK 0.405 62 GLEHDKQET 0.300 273 FNWFVNPLK0.300 262 PLEKPSRPK 0.300 283 FVFFIWRRY 0.300 101 SVWRRSGPF 0.300 140SLELQLPDM 0.300 55 YVKSWVKGL 0.270 27 ELRVVIWNT 0.203 222 FTDMGGNVY0.200 85 WRFVFRFDY 0.180 308 LLVFYTIPG 0.180 198 AQEAQAGKK 0.180 79GEGNFNWRF 0.162 286 FIWRRYWRT 0.150 232 LTGKVEAEF 0.150 295 LVLLLLVLL0.135 11 PAPPPVDIK 0.135 21 RQPISYELR 0.120 170 RCNLFRCRR 0.120 31VIWNTEDVV 0.100 39 VLDDENPLT 0.100 278 NPLKTFVFF 0.090 187 KLKEAEDVE0.090 231 ILTGKVEAE 0.090 265 KPSRPKTSF 0.090 87 FVFRFDYLP 0.090 110ALEEAEFRQ 0.090 307 LLLVFYTIP 0.090 38 VVLDDENPL 0.090 166 GAGPRCNLF0.090 109 FALEEAEFR 0.090 197 EAQEAQAGK 0.090 282 TFVFFIWRR 0.081 179LRGWWPVVK 0.060 257 RKQPEPLEK 0.060 144 QLPDMVRGA 0.060 268 RPKTSFNWF0.060 247 EAEKRPVGK 0.060 2 WIDIFPQDV 0.060 46 LTGEMSSDI 0.045 293RTLVLLLLV 0.045 4 DIFPQDVPA 0.045 77 LTGEGNFNW 0.045 313 TIPGQISQV 0.04593 YLPTEREVS 0.040 230 YILTGKVEA 0.030 76 SLTGEGNFN 0.030 228 NVYILTGKV0.030 57 KSWVKGLEH 0.030 276 FVNPLKTFV 0.030 30 VVIWNTEDV 0.030 199QEAQAGKKK 0.030 69 ETDVHFNSL 0.027 319 SQVIFRPLH 0.027 168 GPRCNLFRC0.027 124 LQVWDYDRI 0.027 96 TEREVSVWR 0.027 24 ISYELRVVI 0.022 159SVQLARNGA 0.020 161 QLARNGAGP 0.020 285 FFIWRRYWR 0.018 250 KRPVGKGRK0.018 214 KGRPEDLEF 0.018 78 TGEGNFNWR 0.018 154 GPELCSVQL 0.018 22QPISYELRV 0.018 Table IX: 158P3D2 v.2a A3-9mers 118 LLVRVYVVK 45.000 57WLNVFPLYR 24.000 46 SLEEEFNHF 9.000 117 KLLVRVYVV 8.100 109 GIPQNRPIK6.000 26 EVKGTVSPK 2.700 162 QLNPIFGEI 1.215 153 DTKERYIPK 0.900 11NLISMVGEI 0.810 219 GLASQYEVW 0.600 182 ELTVAVFEH 0.540 205 DLENRFYSH0.540 90 FLIYPESEA 0.450 191 DLVGSDDLI 0.405 130 LAPADPNGK 0.200 99VLFSEPQIS 0.200 37 VATLKIYNR 0.180 184 TVAVFEHDL 0.180 82 LVGKFKGSF0.180 39 TLKIYNRSL 0.180 119 LVRVYVVKA 0.180 198 LIGETHIDL 0.180 91LIYPESEAV 0.150 165 PIFGEILEL 0.135 228 VQQGPQEPF 0.135 81 HLVGKFKGS0.135 35 KAVATLKIY 0.135 85 KFKGSFLIY 0.108 176 SLPAETELT 0.100 201ETHIDLENR 0.090 98 AVLFSEPQI 0.090 180 ETELTVAVF 0.090 158 YIPKQLNPI0.090 169 EILELSISL 0.081 14 SMVGEIQDQ 0.068 143 VVVSAGRER 0.060 41KIYNRSLEE 0.060 106 ISRGIPQNR 0.045 203 HIDLENRFY 0.040 29 GTVSPKKAV0.034 20 QDQGEAEVK 0.030 27 VKGTVSPKK 0.030 147 AGRERQDTK 0.030 123YVVKATNLA 0.030 129 NLAPADPNG 0.030 170 ILELSISLP 0.030 186 AVFEHDLVG0.030 30 TVSPKKAVA 0.030 84 GKFKGSFLI 0.027 78 GSGHLVGKF 0.027 93YPESEAVLF 0.020 159 IPKQLNPIF 0.020 161 KQLNPIFGE 0.018 100 LFSEPQISR0.018 9 GVNLISMVG 0.018 32 SPKKAVATL 0.018 134 DPNGKADPY 0.018 138KADPYVVVS 0.016 79 SGHLVGKFK 0.015 121 RVYVVKATN 0.015 197 DLIGETHID0.013 77 EGSGHLVGK 0.013 115 PIKLLVRVY 0.012 216 ANCGLASQY 0.012 113NRPIKLLVR 0.012 110 IPQNRPIKL 0.012 53 HFEDWLNVF 0.009 172 ELSISLPAE0.009 56 DWLNVFPLY 0.008 55 EDWLNVFPL 0.008 207 ENRFYSHHR 0.007 45RSLEEEFNH 0.007 175 ISLPAETEL 0.007 222 SQYEVWVQQ 0.007 183 LTVAVFEHD0.007 149 RERQDTKER 0.006 220 LASQYEVWV 0.006 19 IQDQGEAEV 0.006 177LPAETELTV 0.006 5 GDSDGVNLI 0.005 114 RPIKLLVRV 0.005 38 ATLKIYNRS 0.00515 MVGEIQDQG 0.005 88 GSFLIYPES 0.005 155 KERYIPKQL 0.004 36 AVATLKIYN0.004 43 YNRSLEEEF 0.004 124 VVKATNLAP 0.004 192 LVGSDDLIG 0.004 34KKAVATLKI 0.004 163 LNPIFGEIL 0.004 202 THIDLENRF 0.003 33 PKKAVATLK0.003 185 VAVFEHDLV 0.003 52 NHFEDWLNV 0.003 105 QISRGIPQN 0.003 12LISMVGEIQ 0.003 62 PLYRGQGGQ 0.003 174 SISLPAETE 0.003 69 GQDGGGEEE0.003 Table IX: 158P3D2 v.3 A3-9mers 1 PTEREVSVR 0.060 7 SVRRRSGPF 0.0602 TEREVSVRR 0.027 9 RRRSGPFAL 0.002 3 EREVSVRRR 0.000 8 VRRRSGPFA 0.0006 VSVRRRSGP 0.000 5 EVSVRRRSG 0.000 4 REVSVRRRS 0.000 Table IX: 158P3D2v.4 A3-9mers 8 SIWRRSGPF 0.300 3 TEREVSIWR 0.054 1 LPTEREVSI 0.009 4EREVSIWRR 0.005 2 PTEREVSIW 0.003 9 IWRRSGPFA 0.000 6 EVSIWRRSG 0.000 7VSIWRRSGP 0.000 5 REVSIWRRS 0.000 Table IX: 158P3D2 v.5a A3-9mers 16SLDPWSCSY 18.000 2 VLQVWDYTA 1.800 4 QVWDYTASL 0.900 43 AMGPGRGAI 0.27045 GPGRGAICF 0.120 25 QTWCVGPGA 0.075 37 ALCSWPAMG 0.060 11 SLPMTSLDP0.040 14 MTSLDPWSC 0.030 30 GPGAPSSAL 0.027 49 GAICFAAAA 0.027 1LVLQVWDYT 0.022 9 TASLPMTSL 0.013 21 SCSYQTWCV 0.006 28 CVGPGAPSS 0.00612 LPMTSLDPW 0.005 18 DPWSCSYQT 0.005 8 YTASLPMTS 0.004 13 PMTSLDPWS0.004 40 SWPAMGPGR 0.004 33 APSSALCSW 0.003 20 WSCSYQTWC 0.003 35SSALCSWPA 0.003 36 SALCSWPAM 0.003 47 GRGAICFAA 0.003 32 GAPSSALCS 0.0026 WDYTASLPM 0.002 3 LQVWDYTAS 0.002 27 WCVGPGAPS 0.001 38 LCSWPAMGP0.001 48 RGAICFAAA 0.001 24 YQTWCVGPG 0.001 22 CSYQTWCVG 0.001 15TSLDPWSCS 0.000 39 CSWPAMGPG 0.000 29 VGPGAPSSA 0.000 44 MGPGRGAIC 0.00010 ASLPMTSLD 0.000 42 PAMGPGRGA 0.000 23 SYQTWCVGP 0.000 41 WPAMGPGRG0.000 46 PGRGAICFA 0.000 7 DYTASLPMT 0.000 31 PGAPSSALC 0.000 5VWDYTASLP 0.000 17 LDPWSCSYQ 0.000 19 PWSCSYQTW 0.000 34 PSSALCSWP 0.00026 TWCVGPGAP 0.000

[0768] TABLE X 158P3D2 A3, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 1234567890 Score NO. Table X: 158P3D2 v.1 A3-10mers 178 RLRGWWPVVK90.000 281 KTFVFFIWRR 40.500 187 KLKEAEDVER 18.000 241 ELLTVEEAEK 9.000299 LLVLLTVFLL 8.100 302 LLTVFLLLVF 6.000 122 LVLQVWDYDR 5.400 120AVLVLQVWDY 5.400 297 LLLLVLLTVF 4.500 231 ILTGKVEAEF 4.500 242LLTVEEAEKR 4.000 301 VLLTVFLLLV 2.700 144 QLPDMVRGAR 1.800 319SQVIFRPLHK 1.800 296 VLLLLVLLTV 1.350 294 TLVLLLLVLL 1.350 10 VPAPPPVDIK1.350 48 GEMSSDIYVK 1.215 161 QLARNGAGPR 1.200 298 LLLVLLTVFL 0.900 77LTGEGNFNWR 0.900 276 FVNPLKTFVF 0.900 76 SLTGEGNFNW 0.900 300 LVLLTVFLLL0.810 123 VLQVWDYDRI 0.600 303 LTVFLLLVFY 0.450 304 TVFLLLVFYT 0.450 81GNFNWRFVFR 0.360 17 DIKPRQPISY 0.360 166 GAGPRCNLFR 0.360 31 VIWNTEDVVL0.300 107 GPFALEEAEF 0.300 46 LTGEMSSDIY 0.300 198 AQEAQAGKKK 0.300 279PLKTFVFFIW 0.270 278 NPLKTFVFFI 0.243 180 RGWWPVVKLK 0.225 93 YLPTEREVSV0.200 140 SLELQLPDMV 0.200 172 NLFRCRRLRG 0.200 125 QVWDYDRISA 0.200 307LLLVFYTIPG 0.180 235 KVEAEFELLT 0.180 96 TEREVSVWRR 0.162 226 GGNVYILTGK0.135 293 RTLVLLLLVL 0.135 309 LVFYTIPGQI 0.135 224 DMGGNVYILT 0.135 271TSFNWFVNPL 0.135 313 TIPGQISQVI 0.135 256 GRKQPEPLEK 0.120 87 FVFRFDYLPT0.100 101 SVWRRSGPFA 0.100 52 SDIYVKSWVK 0.090 295 LVLLLLVLLT 0.090 148MVRGARGPEL 0.090 306 FLLLVFYTIP 0.090 45 PLTGEMSSDI 0.090 286 FIWRRYWRTL0.090 19 KPRQPISYEL 0.081 280 LKTFVFFIWR 0.072 62 GLEHDKQETD 0.060 259QPEPLEKPSR 0.060 284 VFFIWRRYWR 0.060 196 REAQEAQAGK 0.060 82 NFNWRFVFRF0.054 141 LELQLPDMVR 0.054 121 VLVLQVWDYD 0.045 308 LLVFYTIPGQ 0.045 12APPPVDIKPR 0.045 39 VLDDENPLTG 0.040 84 NWRFVFRFDY 0.036 168 GPRCNLFRCR0.036 117 RQPAVLVLQV 0.036 21 RQPISYELRV 0.036 312 YTIPGQISQV 0.034 272SFNWFVNPLK 0.030 58 SWVKGLEHDK 0.030 200 EAQAGKKKRK 0.030 30 VVIWNTEDVV0.030 283 FVFFIWRRYW 0.030 137 FLGSLELQLP 0.030 222 FTDMGGNVYI 0.030 29RVVIWNTEDV 0.030 95 PTEREVSVWR 0.030 37 DVVLDDENPL 0.027 78 TGEGNFNWRF0.027 9 DVPAPPPVDI 0.027 317 QISQVIFRPL 0.027 270 KTSFNWFVNP 0.027 246EEAEKRPVGK 0.027 197 EAQEAQAGKK 0.027 131 RTSANDFLGS 0.024 24 ISYELRVVIW0.022 261 EPLEKPSRPK 0.020 314 IPGQISQVIF 0.020 202 QAGKKKRKQR 0.020 89FRFDYLPTER 0.020 185 VVKLKEAEDV 0.020 316 GQISQVIFRP 0.018 Table X:158P3D2 v.2a A3-10mers 117 KLLVRVYVVK 135.000 99 VLFSEPQISR 60.000 129NLAPADPNGK 30.000 81 HLVGKFKGSF 4.050 162 QLNPIFGEIL 2.700 118LLVRVYVVKA 2.700 219 GLASQYEVWV 1.800 36 AVATLKIYNR 1.800 26 EVKGTVSPKK1.350 197 DLIGETHIDL 0.810 170 ILELSISLPA 0.600 105 QISRGIPQNR 0.600 19IQDQGEAEVK 0.600 91 LIYPESEAVL 0.450 176 SLPAETELTV 0.400 84 GKFKGSFLIY0.360 109 GIPQNRPIKL 0.360 90 FLIYPESEAV 0.300 121 RVYVVKATNL 0.300 32SPKKAVATLK 0.300 227 WVQQGPQEPF 0.300 25 AEVKGTVSPK 0.270 78 GSGHLVGKFK0.225 158 YIPKQLNPIF 0.200 146 SAGRERQDTK 0.200 205 DLENRFYSHH 0.180 183LTVAVFEHDL 0.135 57 WLNVFPLYRG 0.135 161 KQLNPIFGEI 0.109 46 SLEEEFNHFE0.090 140 DPYVVVSAGR 0.090 45 RSLEEEFNHF 0.068 52 NHFEDWLNVF 0.068 14SMVGEIQDQG 0.068 142 YVVVSAGRER 0.060 82 LVGKFKGSFL 0.060 174 SISLPAETEL0.060 200 GETHIDLENR 0.054 108 RGIPQNRPIK 0.045 41 KIYNRSLEEE 0.045 186AVFEHDLVGS 0.045 11 NLISMVGEIQ 0.045 29 GTVSPKKAVA 0.045 222 SQYEVWVQQG0.041 138 KADPYVVVSA 0.041 215 RANCGLASQY 0.040 152 QDTKERYIPK 0.040 112QNRPIKLLVR 0.036 206 LENRFYSHHR 0.036 172 ELSISLPAET 0.030 201ETHIDLENRF 0.030 124 VVKATNLAPA 0.030 182 ELTVAVFEHD 0.027 164NPIFGEILEL 0.027 76 EEGSGHLVGK 0.027 191 DLVGSDDLIG 0.027 55 EDWLNVFPLY0.027 179 AETELTVAVF 0.027 119 LVRVYVVKAT 0.022 39 TLKIYNRSLE 0.020 184TVAVFEHDLV 0.020 114 RPIKLLVRVY 0.018 168 GEILELSISL 0.016 54 FEDWLNVFPL0.016 30 TVSPKKAVAT 0.015 38 ATLKIYNRSL 0.013 59 NVFPLYRGQG 0.013 203HIDLENRFYS 0.012 149 RERQDTKERY 0.012 56 DWLNVFPLYR 0.011 34 KKAVATLKIY0.009 31 VSPKKAVATL 0.009 9 GVNLISMVGE 0.009 181 TELTVAVFEH 0.008 49EEFNHFEDWL 0.008 165 PIFGEILELS 0.007 110 IPQNRPIKLL 0.007 148GRERQDTKER 0.006 123 YVVKATNLAP 0.006 192 LVGSDDLIGE 0.006 217NCGLASQYEV 0.006 98 AVLFSEPQIS 0.006 18 EIQDQGEAEV 0.006 88 GSFLIYPESE0.005 198 LIGETHIDLE 0.005 177 LPAETELTVA 0.005 194 GSDDLIGETH 0.005 204IDLENRFYSH 0.004 12 LISMVGEIQD 0.004 133 ADPNGKADPY 0.004 92 IYPESEAVLF0.003 15 MVGEIQDQGE 0.003 225 EVWVQQGPQE 0.003 115 PIKLLVRVYV 0.003 62PLYRGQGGQD 0.003 211 YSHHRANCGL 0.003 143 VVVSAGRERQ 0.003 116IKLLVRVYVV 0.003 69 GQDGGGEEEG 0.003 97 EAVLFSEPQI 0.003 Table X:158P3D2 v.3 A3-10mers 1 LPTEREVSVR 0.180 2 PTEREVSVRR 0.030 8 SVRRRSGPFA0.020 3 TEREVSVRRR 0.005 7 VSVRRRSGPF 0.005 9 VRRRSGPFAL 0.002 6EVSVRRRSGP 0.001 10 RRRSGPFALE 0.000 5 REVSVRRRSG 0.000 4 EREVSVRRRS0.000 Table X: 158P3D2 v.4 A3-10mers 1 YLPTEREVSI 0.600 9 SIWRRSGPFA0.100 4 TEREVSIWRR 0.081 3 PTEREVSIWR 0.060 2 LPTEREVSIW 0.009 8VSIWRRSGPF 0.005 10 IWRRSGPFAL 0.002 7 EVSIWRRSGP 0.001 6 REVSIWRRSG0.000 5 EREVSIWRRS 0.000 Table X: 158P3D2 v.5a A3-10mers 44 AMGPGRGAIC0.300 12 SLPMTSLDPW 0.300 2 LVLQVWDYTA 0.270 1 VLVLQVWDYT 0.225 40CSWPAMGPGR 0.150 16 TSLDPWSCSY 0.090 4 LQVWDYTASL 0.081 9 YTASLPMTSL0.068 38 ALCSWPAMGP 0.060 14 PMTSLDPWSC 0.060 3 VLQVWDYTAS 0.040 29CVGPGAPSSA 0.030 17 SLDPWSCSYQ 0.030 5 QVWDYTASLP 0.010 25 YQTWCVGPGA0.009 46 GPGRGAICFA 0.009 33 GAPSSALCSW 0.009 45 MGPGRGAICF 0.006 31GPGAPSSALC 0.006 19 DPWSCSYQTW 0.003 15 MTSLDPWSCS 0.003 21 WSCSYQTWCV0.003 48 GRGAICFAAA 0.002 26 QTWCVGPGAP 0.002 23 CSYQTWCVGP 0.002 30VGPGAPSSAL 0.001 36 SSALCSWPAM 0.001 28 WCVGPGAPSS 0.001 37 SALCSWPAMG0.001 7 WDYTASLPMT 0.001 49 RGAICFAAAA 0.001 13 LPMTSLDPWS 0.001 11ASLPMTSLDP 0.000 43 PAMGPGRGAI 0.000 6 VWDYTASLPM 0.000 18 LDPWSCSYQT0.000 42 WPAMGPGRGA 0.000 35 PSSALCSWPA 0.000 34 APSSALCSWP 0.000 22SCSYQTWCVG 0.000 10 TASLPMTSLD 0.000 47 PGRGAICFAA 0.000 39 LCSWPAMGPG0.000 20 PWSCSYQTWC 0.000 27 TWCVGPGAPS 0.000 8 DYTASLPMTS 0.000 24SYQTWCVGPG 0.000 32 PGAPSSALCS 0.000 41 SWPAMGPGRG 0.000

[0769] TABLE XI 158P3D2 A11, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 123456789 Score NO. Table XI: 158P3D2 v.1 A11-9mers 320 QVIFRPLHK6.000 281 KTFVFFIWR 2.400 59 WVKGLEHDK 2.000 316 GQISQVIFR 1.080 198AQEAQAGKK 0.600 53 DIYVKSWVK 0.480 242 LLTVEEAEK 0.400 21 RQPISYELR0.360 243 LTVEEAEKR 0.300 201 AQAGKKKRK 0.300 49 EMSSDIYVK 0.240 227GNVYILTGK 0.180 123 VLQVWDYDR 0.160 257 RKQPEPLEK 0.120 90 RFDYLPTER0.120 282 TFVFFIWRR 0.120 170 RCNLFRCRR 0.120 285 FFIWRRYWR 0.120 293RTLVLLLLV 0.090 300 LVLLTVFLL 0.090 273 FNWFVNPLK 0.080 181 GWWPVVKLK0.060 250 KRPVGKGRK 0.060 109 FALEEAEFR 0.060 247 EAEKRPVGK 0.060 197EAQEAQAGK 0.060 235 KVEAEFELL 0.060 142 ELQLPDMVR 0.048 156 ELCSVQLAR0.048 145 LPDMVRGAR 0.040 304 TVFLLLVFY 0.040 82 NFNWRFVFR 0.040 101SVWRRSGPF 0.040 228 NVYILTGKV 0.040 162 LARNGAGPR 0.040 295 LVLLLLVLL0.030 77 LTGEGNFNW 0.030 199 QEAQAGKKK 0.030 303 LTVFLLLVF 0.030 38VVLDDENPL 0.030 30 VVIWNTEDV 0.030 290 RYWRTLVLL 0.024 276 FVNPLKTFV0.020 179 LRGWWPVVK 0.020 11 PAPPPVDIK 0.020 159 SVQLARNGA 0.020 172NLFRCRRLR 0.016 204 GKKKRKQRR 0.012 306 FLLLVFYTI 0.012 301 VLLTVFLLL0.012 121 VLVLQVWDY 0.012 96 TEREVSVWR 0.012 178 RLRGWWPVV 0.012 297LLLLVLLTV 0.012 294 TLVLLLLVL 0.012 232 LTGKVEAEF 0.010 222 FTDMGGNVY0.010 55 YVKSWVKGL 0.010 46 LTGEMSSDI 0.010 29 RVVIWNTED 0.009 124LQVWDYDRI 0.009 270 KTSFNWFVN 0.009 86 RFVFRFDYL 0.009 319 SQVIFRPLH0.009 302 LLTVFLLLV 0.008 87 FVFRFDYLP 0.008 137 FLGSLELQL 0.008 167AGPRCNLFR 0.008 31 VIWNTEDVV 0.008 81 GNFNWRFVF 0.007 48 GEMSSDIYV 0.007208 RKQRRRKGR 0.006 206 KKRKQRRRK 0.006 154 GPELCSVQL 0.006 230YILTGKVEA 0.006 22 QPISYELRV 0.006 299 LLVLLTVFL 0.006 193 DVEREAQEA0.006 298 LLLVLLTVF 0.006 265 KPSRPKTSF 0.006 166 GAGPRCNLF 0.006 200EAQAGKKKR 0.006 175 RCRRLRGWW 0.006 268 RPKTSFNWF 0.006 262 PLEKPSRPK0.004 25 SYELRVVIW 0.004 2 WIDIFPQDV 0.004 78 TGEGNFNWR 0.004 188LKEAEDVER 0.004 309 LVFYTIPGQ 0.004 118 QPAVLVLQV 0.004 313 TIPGQISQV0.004 283 FVFFIWRRY 0.004 310 VFYTIPGQI 0.004 140 SLELQLPDM 0.004 131RISANDFLG 0.004 79 GEGNFNWRF 0.004 312 YTIPGQISQ 0.003 278 NPLKTFVFF0.003 120 AVLVLQVWD 0.003 Table XI: 158P3D2 v.2a A11-9mers 109 GIPQNRPIK1.200 153 DTKERYIPK 0.600 118 LLVRVYVVK 0.600 26 EVKGTVSPK 0.600 130LAPADPNGK 0.200 57 WLNVFPLYR 0.160 37 VATLKIYNR 0.080 100 LFSEPQISR0.080 201 ETHIDLENR 0.060 143 VVVSAGRER 0.060 117 KLLVRVYVV 0.036 98AVLFSEPQI 0.030 123 YVVKATNLA 0.030 29 GTVSPKKAV 0.022 27 VKGTVSPKK0.020 30 TVSPKKAVA 0.020 184 TVAVFEHDL 0.020 147 AGRERQDTK 0.020 82LVGKFKGSF 0.020 20 QDQGEAEVK 0.020 119 LVRVYVVKA 0.020 149 RERQDTKER0.018 141 PYVVVSAGR 0.012 85 KFKGSFLIY 0.012 219 GLASQYEVW 0.012 121RVYVVKATN 0.012 9 GVNLISMVG 0.012 79 SGHLVGKFK 0.010 114 RPIKLLVRV 0.009161 KQLNPIFGE 0.008 186 AVFEHDLVG 0.008 113 NRPIKLLVR 0.008 91 LIYPESEAV0.008 198 LIGETHIDL 0.008 228 VQQGPQEPF 0.006 19 IQDQGEAEV 0.006 90FLIYPESEA 0.006 11 NLISMVGEI 0.006 77 EGSGHLVGK 0.006 122 VYVVKATNL0.006 41 KIYNRSLEE 0.005 35 KAVATLKIY 0.005 124 VVKATNLAP 0.004 46SLEEEFNHF 0.004 177 LPAETELTV 0.004 158 YIPKQLNPI 0.004 106 ISRGIPQNR0.004 192 LVGSDDLIG 0.004 36 AVATLKIYN 0.004 110 IPQNRPIKL 0.004 92IYPESEAVL 0.004 162 QLNPIFGEI 0.004 84 GKFKGSFLI 0.004 157 RYIPKQLNP0.004 169 EILELSISL 0.004 182 ELTVAVFEH 0.004 185 VAVFEHDLV 0.003 180ETELTVAVF 0.003 142 YVVVSAGRE 0.003 45 RSLEEEFNH 0.003 17 GEIQDQGEA0.003 207 ENRFYSHHR 0.002 205 DLENRFYSH 0.002 33 PKKAVATLK 0.002 144VVSAGRERQ 0.002 159 IPKQLNPIF 0.002 53 HFEDWLNVF 0.002 32 SPKKAVATL0.002 227 WVQQGPQEP 0.002 131 APADPNGKA 0.002 220 LASQYEVWV 0.002 15MVGEIQDQG 0.002 93 YPESEAVLF 0.002 151 RQDTKERYI 0.002 69 GQDGGGEEE0.002 66 GQGGQDGGG 0.002 23 GEAEVKGTV 0.002 74 GEEEGSGHL 0.002 171LELSISLPA 0.002 191 DLVGSDDLI 0.002 165 PIFGEILEL 0.002 183 LTVAVFEHD0.002 38 ATLKIYNRS 0.002 225 EVWVQQGPQ 0.001 34 KKAVATLKI 0.001 222SQYEVWVQQ 0.001 127 ATNLAPADP 0.001 155 KERYIPKQL 0.001 99 VLFSEPQIS0.001 112 QNRPIKLLV 0.001 52 NHFEDWLNV 0.001 126 KATNLAPAD 0.001 138KADPYVVVS 0.001 78 GSGHLVGKF 0.001 14 SMVGEIQDQ 0.001 218 CGLASQYEV0.001 73 GGEEEGSGH 0.001 215 RANCGLASQ 0.001 164 NPIFGEILE 0.001 5GDSDGVNLI 0.001 Table XI: 158P3D2 v.3 A11-9mers 1 PTEREVSVR 0.020 7SVRRRSGPF 0.020 2 TEREVSVRR 0.012 9 RRRSGPFAL 0.002 8 VRRRSGPFA 0.000 3EREVSVRRR 0.000 5 EVSVRRRSG 0.000 6 VSVRRRSGP 0.000 4 REVSVRRRS 0.000Table XI: 158P3D2 v.4 A11-9mers 3 TEREVSIWR 0.024 8 SIWRRSGPF 0.008 4EREVSIWRR 0.002 1 LPTEREVSI 0.002 2 PTEREVSIW 0.001 9 IWRRSGPFA 0.000 6EVSIWRRSG 0.000 7 VSIWRRSGP 0.000 5 REVSIWRRS 0.000 Table XI: 158P3D2v.5a A11-9mers 4 QVWDYTASL 0.040 25 QTWCVGPGA 0.020 45 GPGRGAICF 0.01249 GAICFAAAA 0.009 2 VLQVWDYTA 0.008 30 GPGAPSSAL 0.006 16 SLDPWSCSY0.004 21 SCSYQTWCV 0.004 43 AMGPGRGAI 0.004 40 SWPAMGPGR 0.004 12LPMTSLDPW 0.004 1 LVLQVWDYT 0.003 36 SALCSWPAM 0.003 14 MTSLDPWSC 0.0029 TASLPMTSL 0.002 33 APSSALCSW 0.002 28 CVGPGAPSS 0.002 8 YTASLPMTS0.002 47 GRGAICFAA 0.002 32 GAPSSALCS 0.001 3 LQVWDYTAS 0.001 11SLPMTSLDP 0.001 6 WDYTASLPM 0.001 24 YQTWCVGPG 0.001 48 RGAICFAAA 0.00137 ALCSWPAMG 0.000 38 LCSWPAMGP 0.000 23 SYQTWCVGP 0.000 35 SSALCSWPA0.000 27 WCVGPGAPS 0.000 18 DPWSCSYQT 0.000 41 WPAMGPGRG 0.000 29VGPGAPSSA 0.000 7 DYTASLPMT 0.000 22 CSYQTWCVG 0.000 13 PMTSLDPWS 0.00039 CSWPAMGPG 0.000 42 PAMGPGRGA 0.000 15 TSLDPWSCS 0.000 10 ASLPMTSLD0.000 26 TWCVGPGAP 0.000 20 WSCSYQTWC 0.000 5 VWDYTASLP 0.000 46PGRGAICFA 0.000 19 PWSCSYQTW 0.000 17 LDPWSCSYQ 0.000 44 MGPGRGAIC 0.00034 PSSALCSWP 0.000 31 PGAPSSALC 0.000

[0770] TABLE XII 158P3D2 A11, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 1234567890 Score NO. Table XII: 158P3D2 v.1 A11-10mers 281KTFVFFIWRR 2.400 319 SQVIFRPLHK 1.800 122 LVLQVWDYDR 1.200 178RLRGWWPVVK 1.200 48 GEMSSDIYVK 0.720 198 AQEAQAGKKK 0.300 166 GAGPRCNLFR0.240 187 KLKEAEDVER 0.240 272 SFNWFVNPLK 0.200 10 VPAPPPVDIK 0.200 77LTGEGNFNWR 0.200 241 ELLTVEEAEK 0.180 196 REAQEAQAGK 0.180 284VFFIWRRYWR 0.160 256 GRKQPEPLEK 0.120 29 RVVIWNTEDV 0.090 293 RTLVLLLLVL0.090 125 QVWDYDRISA 0.080 144 QLPDMVRGAR 0.080 161 QLARNGAGPR 0.080 242LLTVEEAEKR 0.080 226 GGNVYILTGK 0.060 180 RGWWPVVKLK 0.060 52 SDIYVKSWVK0.060 120 AVLVLQVWDY 0.060 300 LVLLTVFLLL 0.060 197 EAQEAQAGKK 0.060 276FVNPLKTFVF 0.060 81 GNFNWRFVFR 0.048 290 RYWRTLVLLL 0.048 101 SVWRRSGPFA0.040 259 QPEPLEKPSR 0.040 309 LVFYTIPGQI 0.040 21 RQPISYELRV 0.036 141LELQLPDMVR 0.036 117 RQPAVLVLQV 0.036 58 SWVKGLEHDK 0.030 200 EAQAGKKKRK0.030 30 VVIWNTEDVV 0.030 96 TEREVSVWRR 0.024 12 APPPVDIKPR 0.020 148MVRGARGPEL 0.020 202 QAGKKKRKQR 0.020 185 VVKLKEAEDV 0.020 95 PTEREVSVWR0.020 299 LLVLLTVFLL 0.018 246 EEAEKRPVGK 0.018 303 LTVFLLLVFY 0.015 312YTIPGQISQV 0.015 168 GPRCNLFRCR 0.012 235 KVEAEFELLT 0.012 19 KPRQPISYEL0.012 304 TVFLLLVFYT 0.012 296 VLLLLVLLTV 0.012 107 GPFALEEAEF 0.012 76SLTGEGNFNW 0.012 301 VLLTVFLLLV 0.012 268 RPKTSFNWFV 0.012 222FTDMGGNVYI 0.010 46 LTGEMSSDIY 0.010 37 DVVLDDENPL 0.009 261 EPLEKPSRPK0.009 278 NPLKTFVFFI 0.009 316 GQISQVIFRP 0.008 280 LKTFVFFIWR 0.008 87FVFRFDYLPT 0.008 302 LLTVFLLLVF 0.008 89 FRFDYLPTER 0.008 31 VIWNTEDVVL0.008 207 KRKQRRRKGR 0.006 205 KKKRKQRRRK 0.006 216 RPEDLEFTDM 0.006 249EKRPVGKGRK 0.006 294 TLVLLLLVLL 0.006 305 VFLLLVFYTI 0.006 82 NFNWRFVFRF0.006 297 LLLLVLLTVF 0.006 199 QEAQAGKKKR 0.006 295 LVLLLLVLLT 0.006 154GPELCSVQLA 0.006 151 GARGPELCSV 0.006 9 DVPAPPPVDI 0.006 298 LLLVLLTVFL0.006 248 AEKRPVGKGR 0.006 229 VYILTGKVEA 0.006 67 KQETDVHFNS 0.005 123VLQVWDYDRI 0.004 93 YLPTEREVSV 0.004 283 FVFFIWRRYW 0.004 203 AGKKKRKQRR0.004 231 ILTGKVEAEF 0.004 108 PFALEEAEFR 0.004 140 SLELQLPDMV 0.004 313TIPGQISQVI 0.004 155 PELCSVQLAR 0.004 38 VVLDDENPLT 0.003 275 WFVNPLKTFV0.003 270 KTSFNWFVNP 0.003 54 IYVKSWVKGL 0.003 131 RISANDFLGS 0.002Table XII: 158P3D2 v.2a A11-10-mers 117 KLLVRVYVVK 1.800 36 AVATLKIYNR0.800 19 IQDQGEAEVK 0.600 26 EVKGTVSPKK 0.600 129 NLAPADPNGK 0.400 99VLFSEPQISR 0.320 32 SPKKAVATLK 0.200 146 SAGRERQDTK 0.200 121 RVYVVKATNL0.120 108 RGIPQNRPIK 0.090 25 AEVKGTVSPK 0.090 105 QISRGIPQNR 0.080 142YVVVSAGRER 0.060 29 GTVSPKKAVA 0.045 152 QDTKERYIPK 0.040 200 GETHIDLENR0.036 78 GSGHLVGKFK 0.030 161 KQLNPIFGEI 0.027 140 DPYVVVSAGR 0.024 109GIPQNRPIKL 0.024 227 WVQQGPQEPF 0.020 82 LVGKFKGSFL 0.020 184 TVAVFEHDLV0.020 124 VVKATNLAPA 0.020 76 EEGSGHLVGK 0.018 157 RYIPKQLNPI 0.018 112QNRPIKLLVR 0.016 183 LTVAVFEHDL 0.015 219 GLASQYEVWV 0.012 206LENRFYSHHR 0.012 170 ILELSISLPA 0.008 176 SLPAETELTV 0.008 91 LIYPESEAVL0.008 148 GRERQDTKER 0.006 164 NPIFGEILEL 0.006 122 VYVVKATNLA 0.006 9GVNLISMVGE 0.006 118 LLVRVYVVKA 0.006 81 HLVGKFKGSF 0.006 138 KADPYVVVSA0.006 215 RANCGLASQY 0.006 123 YVVKATNLAP 0.006 90 FLIYPESEAV 0.006 168GEILELSISL 0.005 59 NVFPLYRGQG 0.004 186 AVFEHDLVGS 0.004 42 IYNRSLEEEF0.004 217 NCGLASQYEV 0.004 166 IFGEILELSI 0.004 192 LVGSDDLIGE 0.004 174SISLPAETEL 0.004 158 YIPKQLNPIF 0.004 162 QLNPIFGEIL 0.004 92 IYPESEAVLF0.004 151 RQDTKERYIP 0.004 197 DLIGETHIDL 0.004 56 DWLNVFPLYR 0.004 143VVVSAGRERQ 0.003 201 ETHIDLENRF 0.003 89 SFLIYPESEA 0.003 98 AVLFSEPQIS0.003 181 TELTVAVFEH 0.003 41 KIYNRSLEEE 0.002 84 GKFKGSFLIY 0.002 130LAPADPNGKA 0.002 30 TVSPKKAVAT 0.002 15 MVGEIQDQGE 0.002 177 LPAETELTVA0.002 66 GQGGQDGGGE 0.002 35 KAVATLKIYN 0.002 69 GQDGGGEEEG 0.002 54FEDWLNVFPL 0.002 74 GEEEGSGHLV 0.002 149 RERQDTKERY 0.002 38 ATLKIYNRSL0.002 203 HIDLENRFYS 0.001 85 KFKGSFLIYP 0.001 209 RFYSHHRANC 0.001 222SQYEVWVQQG 0.001 111 PQNRPIKLLV 0.001 225 EVWVQQGPQE 0.001 18 EIQDQGEAEV0.001 205 DLENRFYSHH 0.001 110 IPQNRPIKLL 0.001 119 LVRVYVVKAT 0.001 127ATNLAPADPN 0.001 45 RSLEEEFNHF 0.001 114 RPIKLLVRVY 0.001 97 EAVLFSEPQI0.001 57 WLNVFPLYRG 0.001 51 FNHFEDWLNV 0.001 12 LISMVGEIQD 0.001 14SMVGEIQDQG 0.001 116 IKLLVRVYVV 0.001 73 GGEEEGSGHL 0.001 44 NRSLEEEFNH0.001 10 VNLISMVGEI 0.001 126 KATNLAPADP 0.001 194 GSDDLIGETH 0.001 83VGKFKGSFLI 0.001 Table XII: 158P3D2 v.3 A11-10mers 1 LPTEREVSVR 0.040 8SVRRRSGPFA 0.020 2 PTEREVSVRR 0.020 3 TEREVSVRRR 0.001 6 EVSVRRRSGP0.001 9 VRRRSGPFAL 0.001 7 VSVRRRSGPF 0.000 10 RRRSGPFALE 0.000 5REVSVRRRSG 0.000 4 EREVSVRRRS 0.000 Table XII: 158P3D2 v.4 A11-10mers 3PTEREVSIWR 0.040 4 TEREVSIWRR 0.024 9 SIWRRSGPFA 0.008 1 YLPTEREVSI0.004 2 LPTEREVSIW 0.002 7 EVSIWRRSGP 0.001 10 IWRRSGPFAL 0.001 8VSIWRRSGPF 0.000 6 REVSIWRRSG 0.000 5 EREVSIWRRS 0.000 Table XII:158P3D2 v.5a A11-10mers 2 LVLQVWDYTA 0.060 29 CVGPGAPSSA 0.020 9YTASLPMTSL 0.010 4 LQVWDYTASL 0.009 40 CSWPAMGPGR 0.008 46 GPGRGAICFA0.006 25 YQTWCVGPGA 0.006 33 GAPSSALCSW 0.006 5 QVWDYTASLP 0.004 12SLPMTSLDPW 0.004 26 QTWCVGPGAP 0.002 19 DPWSCSYQTW 0.001 15 MTSLDPWSCS0.001 38 ALCSWPAMGP 0.001 1 VLVLQVWDYT 0.001 48 GRGAICFAAA 0.001 49RGAICFAAAA 0.001 31 GPGAPSSALC 0.001 45 MGPGRGAICF 0.000 6 VWDYTASLPM0.000 21 WSCSYQTWCV 0.000 43 PAMGPGRGAI 0.000 44 AMGPGRGAIC 0.000 3VLQVWDYTAS 0.000 13 LPMTSLDPWS 0.000 24 SYQTWCVGPG 0.000 17 SLDPWSCSYQ0.000 16 TSLDPWSCSY 0.000 37 SALCSWPAMG 0.000 28 WCVGPGAPSS 0.000 8DYTASLPMTS 0.000 34 APSSALCSWP 0.000 22 SCSYQTWCVG 0.000 10 TASLPMTSLD0.000 30 VGPGAPSSAL 0.000 36 SSALCSWPAM 0.000 42 WPAMGPGRGA 0.000 39LCSWPAMGPG 0.000 14 PMTSLDPWSC 0.000 11 ASLPMTSLDP 0.000 47 PGRGAICFAA0.000 23 CSYQTWCVGP 0.000 7 WDYTASLPMT 0.000 18 LDPWSCSYQT 0.000 35PSSALCSWPA 0.000 41 SWPAMGPGRG 0.000 27 TWCVGPGAPS 0.000 32 PGAPSSALCS0.000 20 PWSCSYQTWC 0.000

[0771] TABLE XIII 158P3D2 A24, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 123456789 Score NO. Table XIII: 158P3D2 v.1 A24-9mers 268RPKTSFNWF 120.000 265 KPSRPKTSF 40.000 278 NPLKTFVFF 20.000 214KGRPEDLEF 9.000 314 IPGQISQVI 8.000 94 LPTEREVSV 8.000 10 VPAPPPVDI8.000 154 GPELCSVQL 6.000 168 GPRCNLFRC 6.000 255 KGRKQPEPL 6.000 133SANDFLGSL 6.000 318 ISQVIFRPL 5.000 75 NSLTGEGNF 5.000 118 QPAVLVLQV4.000 24 ISYELRVVI 4.000 22 QPISYELRV 4.000 38 VVLDDENPL 3.000 55YVKSWVKGL 3.000 175 RCRRLRGWW 3.000 166 GAGPRCNLF 3.000 180 RGWWPVVKL2.000 183 WPVVKLKEA 2.000 283 FVFFIWRRY 2.000 304 TVFLLLVFY 2.000 121VLVLQVWDY 2.000 44 NPLTGEMSS 2.000 19 KPRQPISYE 1.200 178 RLRGWWPVV1.200 299 LLVLLTVFL 1.000 165 NGAGPRCNL 1.000 224 DMGGNVYIL 1.000 277VNPLKTFVF 1.000 298 LLLVLLTVF 1.000 294 TLVLLLLVL 1.000 137 FLGSLELQL1.000 171 CNLFRCRRL 1.000 101 SVWRRSGPF 1.000 81 GNFNWRFVF 1.000 300LVLLTVFLL 1.000 50 MSSDIYVKS 1.000 83 FNWRFVFRF 1.000 232 LTGKVEAEF1.000 303 LTVFLLLVF 1.000 301 VLLTVFLLL 1.000 295 LVLLLLVLL 1.000 77LTGEGNFNW 1.000 235 KVEAEFELL 0.900 151 GARGPELCS 0.900 46 LTGEMSSDI0.800 51 SSDIYVKSW 0.750 132 ISANDFLGS 0.750 222 FTDMGGNVY 0.600 47TGEMSSDIY 0.600 259 QPEPLEKPS 0.600 140 SLELQLPDM 0.600 212 RRKGRPEDL0.600 124 LQVWDYDRI 0.600 293 RTLVLLLLV 0.400 306 FLLLVFYTI 0.400 251RPVGKGRKQ 0.400 6 FPQDVPAPP 0.400 261 EPLEKPSRP 0.400 129 YDRISANDF0.300 291 YWRTLVLLL 0.300 17 DIKPRQPIS 0.300 27 ELRVVIWNT 0.300 287IWRRYWRTL 0.300 69 ETDVHFNSL 0.300 103 WRRSGPFAL 0.300 237 EAEFELLTV0.270 216 RPEDLEFTD 0.240 164 RNGAGPRCN 0.200 234 GKVEAEFEL 0.200 30VVIWNTEDV 0.200 313 TIPGQISQV 0.200 18 IKPRQPISY 0.200 150 RGARGPELC0.200 297 LLLLVLLTV 0.200 42 DENPLTGEM 0.200 107 GPFALEEAE 0.200 290RYWRTLVLL 0.200 302 LLTVFLLLV 0.200 12 APPPVDIKP 0.200 31 VIWNTEDVV0.200 276 FVNPLKTFV 0.200 228 NVYILTGKV 0.200 125 QVWDYDRIS 0.200 86RFVFRFDYL 0.200 144 QLPDMVRGA 0.200 66 DKQETDVHF 0.200 80 EGNFNWRFV0.200 85 WRFVFRFDY 0.200 289 RRYWRTLVL 0.200 270 KTSFNWFVN 0.200 113EAEFRQPAV 0.180 190 EAEDVEREA 0.180 76 SLTGEGNFN 0.150 266 PSRPKTSFN0.150 32 IWNTEDVVL 0.150 119 PAVLVLQVW 0.150 Table XIII: 158P3D2 v.2aA24-9mers 92 IYPESEAVL 360.000 122 VYVVKATNL 300.000 50 EFNHFEDWL 30.00053 HFEDWLNVF 21.600 169 EILELSISL 8.640 175 ISLPAETEL 7.920 110IPQNRPIKL 6.600 163 LNPIFGEIL 6.000 46 SLEEEFNHF 5.184 210 FYSHHRANC5.000 198 LIGETHIDL 4.800 83 VGKFKGSFL 4.000 32 SPKKAVATL 4.000 39TLKIYNRSL 4.000 184 TVAVFEHDL 4.000 108 RGIPQNRPI 3.600 162 QLNPIFGEI3.326 228 VQQGPQEPF 3.000 180 ETELTVAVF 3.000 93 YPESEAVLF 3.000 43YNRSLEEEF 2.640 78 GSGHLVGKF 2.640 159 IPKQLNPIF 2.400 82 LVGKFKGSF2.000 151 RQDTKERYI 2.000 167 FGEILELSI 1.800 157 RYIPKQLNP 1.800 158YIPKQLNPI 1.800 11 NLISMVGEI 1.650 191 DLVGSDDLI 1.500 98 AVLFSEPQI1.500 85 KFKGSFLIY 1.200 155 KERYIPKQL 1.120 209 RFYSHHRAN 1.000 223QYEVWVQQG 0.900 166 IFGEILELS 0.840 42 IYNRSLEEE 0.825 187 VFEHDLVGS0.750 74 GEEEGSGHL 0.720 190 HDLVGSDDL 0.600 111 PQNRPIKLL 0.600 202THIDLENRF 0.518 63 LYRGQGGQD 0.500 165 PIFGEILEL 0.440 4 PGDSDGVNL 0.40055 EDWLNVFPL 0.400 212 SHHRANCGL 0.400 114 RPIKLLVRV 0.360 117 KLLVRVYVV0.300 35 KAVATLKIY 0.300 121 RVYVVKATN 0.280 38 ATLKIYNRS 0.252 56DWLNVFPLY 0.252 138 KADPYVVVS 0.240 34 KKAVATLKI 0.220 28 KGTVSPKKA0.220 102 SEPQISRGI 0.210 173 LSISLPAET 0.198 81 HLVGKFKGS 0.180 123YVVKATNLA 0.180 8 DGVNLISMV 0.180 112 QNRPIKLLV 0.168 218 CGLASQYEV0.165 90 FLIYPESEA 0.165 194 GSDDLIGET 0.158 88 GSFLIYPES 0.154 31VSPKKAVAT 0.150 185 VAVFEHDLV 0.150 24 EAEVKGTVS 0.150 29 GTVSPKKAV0.150 196 DDLIGETHI 0.150 134 DPNGKADPY 0.150 176 SLPAETELT 0.150 128TNLAPADPN 0.150 22 QGEAEVKGT 0.150 5 GDSDGVNLI 0.144 6 DSDGVNLIS 0.140131 APADPNGKA 0.132 36 AVATLKIYN 0.120 99 VLFSEPQIS 0.120 30 TVSPKKAVA0.120 146 SAGRERQDT 0.120 91 LIYPESEAV 0.120 177 LPAETELTV 0.120 3DPGDSDGVN 0.120 216 ANCGLASQY 0.120 119 LVRVYVVKA 0.110 19 IQDQGEAEV0.110 141 PYVVVSAGR 0.105 220 LASQYEVWV 0.100 136 NGKADPYVV 0.100 51FNHFEDWLN 0.100 84 GKFKGSFLI 0.100 219 GLASQYEVW 0.100 71 DGGGEEEGS0.100 105 QISRGIPQN 0.100 203 HIDLENRFY 0.100 89 SFLIYPESE 0.075 60VFPLYRGQG 0.075 100 LFSEPQISR 0.060 Table XIII: 158P3D2 v.3 A24-9mers 7SVRRRSGPF 2.000 9 RRRSGPFAL 0.800 4 REVSVRRRS 0.042 6 VSVRRRSGP 0.015 8VRRRSGPFA 0.010 5 EVSVRRRSG 0.010 2 TEREVSVRR 0.002 3 EREVSVRRR 0.002 1PTEREVSVR 0.002 Table XIII: 158P3D2 v.4 A24-9mers 8 SIWRRSGPF 2.000 1LPTEREVSI 1.200 9 IWRRSGPFA 0.100 5 REVSIWRRS 0.042 7 VSIWRRSGP 0.015 2PTEREVSIW 0.015 6 EVSIWRRSG 0.010 3 TEREVSIWR 0.002 4 EREVSIWRR 0.002Table XIII: 158P3D2 v.5a A24-9mers 7 DYTASLPMT 5.000 4 QVWDYTASL 4.80030 GPGAPSSAL 4.000 9 TASLPMTSL 4.000 45 GPGRGAICF 2.000 43 AMGPGRGAI1.200 23 SYQTWCVGP 0.750 36 SALCSWPAM 0.750 48 RGAICFAAA 0.240 1LVLQVWDYT 0.210 15 TSLDPWSCS 0.180 29 VGPGAPSSA 0.150 27 WCVGPGAPS 0.1503 LQVWDYTAS 0.150 12 LPMTSLDPW 0.150 32 GAPSSALCS 0.150 49 GAICFAAAA0.150 44 MGPGRGAIC 0.150 2 VLQVWDYTA 0.150 25 QTWCVGPGA 0.140 8YTASLPMTS 0.120 16 SLDPWSCSY 0.120 28 CVGPGAPSS 0.120 14 MTSLDPWSC 0.10033 APSSALCSW 0.100 35 SSALCSWPA 0.100 21 SCSYQTWCV 0.100 18 DPWSCSYQT0.100 20 WSCSYQTWC 0.100 6 WDYTASLPM 0.050 10 ASLPMTSLD 0.018 40SWPAMGPGR 0.015 11 SLPMTSLDP 0.015 42 PAMGPGRGA 0.015 47 GRGAICFAA 0.01419 PWSCSYQTW 0.012 13 PMTSLDPWS 0.012 31 PGAPSSALC 0.012 39 CSWPAMGPG0.012 24 YQTWCVGPG 0.010 41 WPAMGPGRG 0.010 5 VWDYTASLP 0.010 22CSYQTWCVG 0.010 46 PGRGAICFA 0.010 37 ALCSWPAMG 0.010 26 TWCVGPGAP 0.01038 LCSWPAMGP 0.010 17 LDPWSCSYQ 0.002 34 PSSALCSWP 0.001

[0772] TABLE XIV 158P3D2 A24, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 1234567890 Score NO. Table XIV: 158P3D2 v.1 A24-10mers 290RYWRTLVLLL 480.000 54 IYVKSWVKGL 300.000 128 DYDRISANDF 120.000 136DFLGSLELQL 36.000 115 EFRQPAVLVL 20.000 82 NFNWRFVFRF 15.000 153RGPELCSVQL 14.400 293 RTLVLLLLVL 14.400 305 VFLLLVFYTI 12.600 19KPRQPISYEL 12.320 170 RCNLFRCRRL 12.000 25 SYELRVVIWN 10.500 300LVLLTVFLLL 10.080 92 DYLPTEREVS 9.000 229 VYILTGKVEA 8.250 164RNGAGPRCNL 8.000 37 DVVLDDENPL 7.200 294 TLVLLLLVLL 7.200 298 LLLVLLTVFL7.200 317 QISQVIFRPL 6.720 299 LLVLLTVFLL 6.000 113 EAEFRQPAVL 6.000 291YWRTLVLLLL 5.600 271 TSFNWFVNPL 4.800 134 ANDFLGSLEL 4.400 233TGKVEAEFEL 4.400 148 MVRGARGPEL 4.400 31 VIWNTEDVVL 4.000 286 FIWRRYWRTL4.000 132 ISANDFLGSL 4.000 102 VWRRSGPFAL 4.000 277 VNPLKTFVFF 3.600 276FVNPLKTFVF 3.600 297 LLLLVLLTVF 3.600 231 ILTGKVEAEF 3.080 80 EGNFNWRFVF3.000 78 TGEGNFNWRF 3.000 100 VSVWRRSGPF 3.000 313 TIPGQISQVI 2.520 302LLTVFLLLVF 2.400 165 NGAGPRCNLF 2.400 107 GPFALEEAEF 2.200 216RPEDLEFTDM 2.160 314 IPGQISQVIF 2.000 74 FNSLTGEGNF 2.000 274 NWFVNPLKTF2.000 9 DVPAPPPVDI 1.500 278 NPLKTFVFFI 1.500 123 VLQVWDYDRI 1.500 309LVFYTJPGQI 1.400 282 TFVFFIWRRY 1.050 222 FTDMGGNVYI 1.000 275WFVNPLKTFV 0.900 139 GSLELQLPDM 0.900 234 GKVEAEFELL 0.864 239EFELLTVEEA 0.825 211 RRRKGRPEDL 0.800 289 RRYWRTLVLL 0.800 285FFIWRRYWRT 0.750 73 HFNSLTGEGN 0.750 221 EFTDMGGNVY 0.720 68 QETDVHFNSL0.691 223 TDMGGNVYIL 0.600 310 VFYTIPGQIS 0.600 311 FYTIPGQISQ 0.500 173LFRCRRLRGW 0.500 85 WRFVFRFDYL 0.480 61 KGLEHDKQET 0.475 213 RKGRPEDLEF0.440 179 LRGWWPVVKL 0.440 267 SRPKTSFNWF 0.432 258 KQPEPLEKPS 0.432 67KQETDVHFNS 0.420 129 YDRISANDFL 0.400 254 GKGRKQPEPL 0.400 288WRRYWRTLVL 0.400 117 RQPAVLVLQV 0.360 29 RVVIWNTEDV 0.300 235 KVEAEFELLT0.300 264 EKPSRPKTSF 0.300 21 RQPISYELRV 0.300 105 RSGPFALEEA 0.264 214KGRPEDLEFT 0.240 131 RISANDFLGS 0.240 1 MWIDIFPQDV 0.216 296 VLLLLVLLTV0.210 268 RPKTSFNWFV 0.200 265 KPSRPKTSFN 0.200 65 HDKQETDVHF 0.200 150RGARGPELCS 0.200 227 GNVYILTGKV 0.198 154 GPELCSVQLA 0.180 303LTVFLLLVFY 0.180 38 VVLDDENPLT 0.180 312 YTIPGQISQV 0.180 158 CSVQLARNGA0.180 143 LQLPDMVRGA 0.180 295 LVLLLLVLLT 0.180 244 TVEEAEKRPV 0.180 75NSLTGEGNFN 0.180 Table XIV: 158P3D2 v.2a A24-10mers 157 RYIPKQLNPI216.000 42 IYNRSLEEEF 198.000 92 IYPESEAVLF 180.000 45 RSLEEEFNHF 10.368122 VYVVKATNLA 9.000 121 RVYVVKATNL 8.000 166 IFGEILELSI 7.200 73GGEEEGSGHL 7.200 162 QLNPIFGEIL 7.200 164 NPIFGEILEL 6.600 109GIPQNRPIKL 6.600 31 VSPKKAVATL 6.000 38 ATLKIYNRSL 6.000 110 IPQNRPIKLL6.000 183 LTVAVFEHDL 6.000 197 DLIGETHIDL 6.000 161 KQLNPIFGEI 5.544 3DPGDSDGVNL 4.800 91 LIYPESEAVL 4.800 174 SISLPAETEL 4.400 211 YSHHRANCGL4.000 82 LVGKFKGSFL 4.000 158 YIPKQLNPIF 3.600 227 WVQQGPQEPF 3.000 81HLVGKFKGSF 3.000 201 ETHIDLENRF 2.880 77 EGSGHLVGKF 2.640 101 FSEPQISRGI2.520 10 VNLISMVGEI 1.650 97 EAVLFSEPQI 1.500 223 QYEVWVQQGP 1.260 209RFYSHHRANC 1.000 83 VGKFKGSFLI 1.000 154 TKERYIPKQL 0.840 89 SFLIYPESEA0.825 50 EFNHFEDWLN 0.750 168 GEILELSISL 0.720 63 LYRGQGGQDG 0.600 210FYSHHRANCG 0.600 6 DSDGVNLISM 0.500 189 EHDLVGSDDL 0.400 49 EEFNHFEDWL0.400 54 FEDWLNVFPL 0.400 114 RPIKLLVRVY 0.360 215 RANCGLASQY 0.360 35KAVATLKIYN 0.360 138 KADPYVVVSA 0.336 87 KGSFLIYPES 0.308 52 NHFEDWLNVF0.288 179 AETELTVAVF 0.240 199 IGETHIDLEN 0.231 22 QGEAEVKGTV 0.210 170ILELSISLPA 0.210 28 KGTVSPKKAV 0.200 18 EIQDQGEAEV 0.198 150 ERQDTKERYI0.180 175 ISLPAETELT 0.180 98 AVLFSEPQIS 0.180 37 VATLKIYNRS 0.168 130LAPADPNGKA 0.165 16 VGEIQDQGEA 0.165 118 LLVRVYVVKA 0.165 193 VGSDDLIGET0.158 167 FGEILELSIS 0.150 90 FLIYPESEAV 0.150 29 GTVSPKKAVA 0.150 93YPESEAVLFS 0.150 190 HDLVGSDDLI 0.150 218 CGLASQYEVW 0.150 127ATNLAPADPN 0.150 176 SLPAETELTV 0.150 134 DPNGKADPYV 0.150 119LVRVYVVKAT 0.140 172 ELSISLPAET 0.132 30 TVSPKKAVAT 0.120 145 VSAGRERQDT0.120 4 PGDSDGVNLI 0.120 21 DQGEAEVKGT 0.120 186 AVFEHDLVGS 0.120 177LPAETELTVA 0.120 217 NCGLASQYEV 0.110 53 HFEDWLNVFP 0.108 107 SRGIPQNRPI0.100 184 TVAVFEHDLV 0.100 124 VVKATNLAPA 0.100 207 ENRFYSHHRA 0.100 203HIDLENRFYS 0.100 136 NGKADPYVVV 0.100 51 FNHFEDWLNV 0.100 85 KFKGSFLIYP0.100 195 SDDLIGETHI 0.100 219 GLASQYEVWV 0.100 43 YNRSLEEEFN 0.100 60VFPLYRGQGG 0.090 187 VFEHDLVGSD 0.090 141 PYVVVSAGRE 0.075 100LFSEPQISRG 0.060 117 KLLVRVYVVK 0.042 108 RGIPQNRPIK 0.036 65 RGQGGQDGGG0.030 Table XIV: 158P3D2 v.3 A24-10mers 7 VSVRRRSGPF 3.000 9 VRRRSGPFAL0.400 8 SVRRRSGPFA 0.100 4 EREVSVRRRS 0.021 1 LPTEREVSVR 0.012 6EVSVRRRSGP 0.010 5 REVSVRRRSG 0.003 10 RRRSGPFALE 0.002 2 PTEREVSVRR0.002 3 TEREVSVRRR 0.001 Table XIV: 158P3D2 v.4 A24-10mers 10 IWRRSGPFAL4.000 8 VSIWRRSGPF 3.000 1 YLPTEREVSI 1.500 2 LPTEREVSIW 0.120 9SIWRRSGPFA 0.100 5 EREVSIWRRS 0.021 7 EVSIWRRSGP 0.010 6 REVSIWRRSG0.003 3 PTEREVSIWR 0.002 4 TEREVSIWRR 0.001 Table XIV: 158P3D2 v.5aA24-10mers 8 DYTASLPMTS 6.000 4 LQVWDYTASL 6.000 30 VGPGAPSSAL 6.000 9YTASLPMTSL 4.000 45 MGPGRGAICF 3.000 24 SYQTWCVGPG 0.750 36 SSALCSWPAM0.500 6 VWDYTASLPM 0.500 1 VLVLQVWDYT 0.210 49 RGAICFAAAA 0.200 16TSLDPWSCSY 0.180 13 LPMTSLDPWS 0.180 43 PAMGPGRGAI 0.150 3 VLQVWDYTAS0.150 12 SLPMTSLDPW 0.150 28 WCVGPGAPSS 0.150 33 GAPSSALCSW 0.150 2LVLQVWDYTA 0.150 25 YQTWCVGPGA 0.140 19 DPWSCSYQTW 0.120 29 CVGPGAPSSA0.120 44 AMGPGRGAIC 0.120 27 TWCVGPGAPS 0.100 42 WPAMGPGRGA 0.100 15MTSLDPWSCS 0.100 46 GPGRGAICFA 0.100 21 WSCSYQTWCV 0.100 31 GPGAPSSALC0.100 11 ASLPMTSLDP 0.018 18 LDPWSCSYQT 0.015 37 SALCSWPAMG 0.015 41SWPAMGPGRG 0.015 47 PGRGAICFAA 0.014 34 APSSALCSWP 0.012 40 CSWPAMGPGR0.012 48 GRGAICFAAA 0.012 32 PGAPSSALCS 0.012 17 SLDPWSCSYQ 0.012 5QVWDYTASLP 0.012 7 WDYTASLPMT 0.010 39 LCSWPAMGPG 0.010 23 CSYQTWCVGP0.010 20 PWSCSYQTWC 0.010 14 PMTSLDPWSC 0.010 26 QTWCVGPGAP 0.010 10TASLPMTSLD 0.010 35 PSSALCSWPA 0.010 38 ALCSWPAMGP 0.010 22 SCSYQTWCVG0.010

[0773] TABLE XV 158P3D2 B7, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. IDPos 123456789 Score NO. Table XV: 158P3D2 v.1 B7-9mers 255 KGRKQPEPL40.000 154 GPELCSVQL 24.000 300 LVLLTVFLL 20.000 55 YVKSWVKGL 20.000 168GPRCNLFRC 20.000 295 LVLLLLVLL 20.000 38 VVLDDENPL 20.000 133 SANDFLGSL12.000 10 VPAPPPVDI 12.000 165 NGAGPRCNL 9.000 314 IPGQISQVI 8.000 180RGWWPVVKL 6.000 235 KVEAEFELL 6.000 294 TLVLLLLVL 4.000 94 LPTEREVSV4.000 22 QPISYELRV 4.000 301 VLLTVFLLL 4.000 291 YWRTLVLLL 4.000 318ISQVIFRPL 4.000 103 WRRSGPFAL 4.000 299 LLVLLTVFL 4.000 118 QPAVLVLQV4.000 137 FLGSLELQL 4.000 287 IWRRYWRTL 4.000 171 CNLFRCRRL 4.000 224DMGGNVYIL 4.000 19 KPRQPISYE 3.000 178 RLRGWWPVV 2.000 183 WPVVKLKEA2.000 114 AEFRQPAVL 1.200 69 ETDVHFNSL 1.200 276 FVNPLKTFV 1.000 27ELRVVIWNT 1.000 30 VVIWNTEDV 1.000 228 NVYILTGKV 1.000 151 GARGPELCS0.900 159 SVQLARNGA 0.750 148 MVRGARGPE 0.750 24 ISYELRVVI 0.600 265KPSRPKTSF 0.600 12 APPPVDIKP 0.600 292 WRTLVLLLL 0.400 32 IWNTEDVVL0.400 289 RRYWRTLVL 0.400 149 VRGARGPEL 0.400 46 LTGEMSSDI 0.400 306FLLLVFYTI 0.400 272 SFNWFVNPL 0.400 234 GKVEAEFEL 0.400 278 NPLKTFVFF0.400 130 DRISANDFL 0.400 86 RFVFRFDYL 0.400 135 NDFLGSLEL 0.400 44NPLTGEMSS 0.400 212 RRKGRPEDL 0.400 268 RPKTSFNWF 0.400 290 RYWRTLVLL0.400 116 FRQPAVLVL 0.400 124 LQVWDYDRI 0.400 140 SLELQLPDM 0.300 288WRRYWRTLV 0.300 162 LARNGAGPR 0.300 115 EFRQPAVLV 0.300 175 RCRRLRGWW0.300 214 KGRPEDLEF 0.200 80 EGNFNWRFV 0.200 302 LLTVFLLLV 0.200 297LLLLVLLTV 0.200 261 EPLEKPSRP 0.200 107 GPFALEEAE 0.200 31 VIWNTEDVV0.200 313 TIPGQISQV 0.200 251 RPVGKGRKQ 0.200 293 RTLVLLLLV 0.200 6FPQDVPAPP 0.200 237 EAEFELLTV 0.180 113 EAEFRQPAV 0.180 193 DVEREAQEA0.150 120 AVLVLQVWD 0.150 259 QPEPLEKPS 0.120 223 TDMGGNVYI 0.120 283FVFFIWRRY 0.100 106 SGPFALEEA 0.100 101 SVWRRSGPF 0.100 150 RGARGPELC0.100 304 TVFLLLVFY 0.100 42 DENPLTGEM 0.100 296 VLLLLVLLT 0.100 125QVWDYDRIS 0.100 225 MGGNVYILT 0.100 144 QLPDMVRGA 0.100 102 VWRRSGPFA0.100 4 DIFPQDVPA 0.100 88 VFRFDYLPT 0.100 209 KQRRRKGRP 0.100 286FIWRRYWRT 0.100 230 YILTGKVEA 0.100 16 VDIKPRQPI 0.090 145 LPDMVRGAR0.090 190 EAEDVEREA 0.090 Table XV: 158P3D2 v.2a B7-9-mers 32 SPKKAVATL80.000 110 IPQNRPIKL 80.000 184 TVAVFEHDL 20.000 131 APADPNGKA 9.000 98AVLFSEPQI 6.000 119 LVRVYVVKA 5.000 198 LIGETHIDL 4.000 175 ISLPAETEL4.000 169 EILELSISL 4.000 114 RPIKLLVRV 4.000 83 VGKFKGSFL 4.000 163LNPIFGEIL 4.000 39 TLKIYNRSL 4.000 177 LPAETELTV 4.000 155 KERYIPKQL4.000 112 QNRPIKLLV 2.000 220 LASQYEVWV 0.600 111 PQNRPIKLL 0.600 185VAVFEHDLV 0.600 123 YVVKATNLA 0.500 30 TVSPKKAVA 0.500 146 SAGRERQDT0.450 190 HDLVGSDDL 0.400 92 IYPESEAVL 0.400 212 SHHRANCGL 0.400 122VYVVKATNL 0.400 55 EDWLNVFPL 0.400 191 DLVGSDDLI 0.400 165 PIFGEILEL0.400 50 EFNHFEDWL 0.400 159 IPKQLNPIF 0.400 108 RGIPQNRPI 0.400 162QLNPIFGEI 0.400 134 DPNGKADPY 0.400 3 DPGDSDGVN 0.400 11 NLISMVGEI 0.400158 YIPKQLNPI 0.400 29 GTVSPKKAV 0.300 103 EPQISRGIP 0.300 147 AGRERQDTK0.300 36 AVATLKIYN 0.300 61 FPLYRGQGG 0.200 164 NPIFGEILE 0.200 136NGKADPYVV 0.200 8 DGVNLISMV 0.200 218 CGLASQYEV 0.200 43 YNRSLEEEF 0.200117 KLLVRVYVV 0.200 91 LIYPESEAV 0.200 140 DPYVVVSAG 0.200 186 AVFEHDLVG0.150 90 FLIYPESEA 0.150 93 YPESEAVLF 0.120 74 GEEEGSGHL 0.120 167FGEILELSI 0.120 151 RQDTKERYI 0.120 4 PGDSDGVNL 0.120 207 ENRFYSHHR0.100 28 KGTVSPKKA 0.100 213 HHRANCGLA 0.100 173 LSISLPAET 0.100 176SLPAETELT 0.100 7 SDGVNLISM 0.100 31 VSPKKAVAT 0.100 121 RVYVVKATN 0.100106 ISRGIPQNR 0.100 82 LVGKFKGSF 0.100 144 VVSAGRERQ 0.075 38 ATLKIYNRS0.060 179 AETELTVAV 0.060 216 ANCGLASQY 0.060 35 KAVATLKIY 0.060 19IQDQGEAEV 0.060 143 VVVSAGRER 0.050 192 LVGSDDLIG 0.050 142 YVVVSAGRE0.050 9 GVNLISMVG 0.050 124 VVKATNLAP 0.050 59 NVFPLYRGQ 0.050 26EVKGTVSPK 0.050 225 EVWVQQGPQ 0.050 227 WVQQGPQEP 0.050 15 MVGEIQDQG0.050 34 KKAVATLKI 0.040 5 GDSDGVNLI 0.040 196 DDLIGETHI 0.040 84GKFKGSFLI 0.040 102 SEPQISRGI 0.040 116 IKLLVRVYV 0.030 128 TNLAPADPN0.030 126 KATNLAPAD 0.030 221 ASQYEVWVQ 0.030 130 LAPADPNGK 0.030 228VQQGPQEPF 0.030 37 VATLKIYNR 0.030 137 GKADPYVVV 0.030 139 ADPYVVVSA0.030 13 ISMVGEIQD 0.030 215 RANCGLASQ 0.030 127 ATNLAPADP 0.030 TableXV: 158P3D2 v.3 B7-9mers 9 RRRSGPFAL 4.000 7 SVRRRSGPF 1.000 8 VRRRSGPFA0.100 5 EVSVRRRSG 0.075 6 VSVRRRSGP 0.015 2 TEREVSVRR 0.010 4 REVSVRRRS0.003 3 EREVSVRRR 0.000 1 PTEREVSVR 0.000 Table XV: 158P3D2 v.4 B7-9mers1 LPTEREVSI 8.000 9 IWRRSGPFA 0.100 6 EVSIWRRSG 0.075 8 SIWRRSGPF 0.0207 VSIWRRSGP 0.015 3 TEREVSIWR 0.010 5 REVSIWRRS 0.002 2 PTEREVSIW 0.0014 EREVSIWRR 0.000 Table XV: 158P3D2 v.5a -B7-9-mers 30 GPGAPSSAL 120.0004 QVWDYTASL 20.000 9 TASLPMTSL 18.000 36 SALCSWPAM 3.000 18 DPWSCSYQT2.000 43 AMGPGRGAI 1.800 12 LPMTSLDPW 1.200 33 APSSALCSW 1.200 1LVLQVWDYT 0.500 45 GPGRGAICF 0.400 49 GAICFAAAA 0.300 41 WPAMGPGRG 0.20021 SCSYQTWCV 0.200 42 PAMGPGRGA 0.135 48 RGAICFAAA 0.100 6 WDYTASLPM0.100 2 VLQVWDYTA 0.100 35 SSALCSWPA 0.100 28 CVGPGAPSS 0.100 46PGRGAICFA 0.100 20 WSCSYQTWC 0.100 29 VGPGAPSSA 0.100 25 QTWCVGPGA 0.10014 MTSLDPWSC 0.100 44 MGPGRGAIC 0.100 32 GAPSSALCS 0.060 15 TSLDPWSCS0.030 27 WCVGPGAPS 0.030 10 ASLPMTSLD 0.030 37 ALCSWPAMG 0.030 8YTASLPMTS 0.020 3 LQVWDYTAS 0.020 38 LCSWPAMGP 0.015 11 SLPMTSLDP 0.01031 PGAPSSALC 0.010 22 CSYQTWCVG 0.010 39 CSWPAMGPG 0.010 47 GRGAICFAA0.010 24 YQTWCVGPG 0.010 7 DYTASLPMT 0.010 16 SLDPWSCSY 0.006 13PMTSLDPWS 0.002 17 LDPWSCSYQ 0.001 40 SWPAMGPGR 0.001 23 SYQTWCVGP 0.00126 TWCVGPGAP 0.001 34 PSSALCSWP 0.001 5 VWDYTASLP 0.000 19 PWSCSYQTW0.000

[0774] TABLE XVI 158P3D2 B7, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 1234567890 Score NO. Table XVI: 158P3D2 v.1 B7-10mers 19KPRQPISYEL 800.000 148 MVRGARGPEL 200.000 37 DVVLDDENPL 20.000 300LVLLTVFLLL 20.000 164 RNGAGPRCNL 9.000 278 NPLKTFVFFI 8.000 151GARGPELCSV 6.000 216 RPEDLEFTDM 6.000 31 VIWNTEDVVL 4.000 298 LLLVLLTVFL4.000 294 TLVLLLLVLL 4.000 129 YDRISANDFL 4.000 132 ISANDFLGSL 4.000 288WRRYWRTLVL 4.000 170 RCNLFRCRRL 4.000 22 QPISYELRVV 4.000 115 EFRQPAVLVL4.000 153 RGPELCSVQL 4.000 293 RTLVLLLLVL 4.000 291 YWRTLVLLLL 4.000 286FIWRRYWRTL 4.000 271 TSFNWFVNPL 4.000 233 TGKVEAEFEL 4.000 268RPKTSFNWFV 4.000 299 LLVLLTVFLL 4.000 211 RRRKGRPEDL 4.000 102VWRRSGPFAL 4.000 317 QISQVIFRPL 4.000 134 ANDFLGSLEL 3.600 113EAEFRQPAVL 3.600 9 DVPAPPPVDI 3.000 162 LARNGAGPRC 3.000 309 LVFYTIPGQI2.000 168 GPRCNLFRCR 2.000 223 TDMGGNVYIL 1.200 30 VVIWNTEDVV 1.000 29RVVIWNTEDV 1.000 214 KGRPEDLEFT 1.000 185 VVKLKEAEDV 1.000 139GSLELQLPDM 1.000 125 QVWDYDRISA 0.750 12 APPPVDIKPR 0.600 154 GPELCSVQLA0.600 179 LRGWWPVVKL 0.600 295 LVLLLLVLLT 0.500 38 VVLDDENPLT 0.500 87FVFRFDYLPT 0.500 101 SVWRRSGPFA 0.500 304 TVFLLLVFYT 0.500 54 IYVKSWVKGL0.400 313 TIPGQISQVI 0.400 289 RRYWRTLVLL 0.400 136 DFLGSLELQL 0.400 234GKVEAEFELL 0.400 254 GKGRKQPEPL 0.400 118 QPAVLVLQVW 0.400 314IPGQISQVIF 0.400 68 QETDVHFNSL 0.400 107 GPFALEEAEF 0.400 123 VLQVWDYDRI0.400 290 RYWRTLVLLL 0.400 94 LPTEREVSVW 0.400 265 KPSRPKTSFN 0.400 85WRFVFRFDYL 0.400 261 EPLEKPSRPK 0.300 10 VPAPPPVDIK 0.300 120 AVLVLQVWDY0.300 167 AGPRCNLFRC 0.300 287 IWRRYWRTLV 0.300 244 TVEEAEKRPV 0.300 251RPVGKGRKQP 0.300 6 FPQDVPAPPP 0.300 296 VLLLLVLLTV 0.200 117 RQPAVLVLQV0.200 44 NPLTGEMSSD 0.200 176 CRRLRGWWPV 0.200 183 WPVVKLKEAE 0.200 301VLLTVFLLLV 0.200 227 GNVYILTGKV 0.200 21 RQPISYELRV 0.200 312 YTIPGQISQV0.200 93 YLPTEREVSV 0.200 235 KVEAEFELLT 0.150 158 CSVQLARNGA 0.150 283FVFFIWRRYW 0.150 255 KGRKQPEPLE 0.150 15 PVDIKPRQPI 0.135 222 FTDMGGNVYI0.120 209 KQRRRKGRPE 0.100 105 RSGPFALEEA 0.100 27 ELRVVIWNTE 0.100 273FNWFVNPLKT 0.100 143 LQLPDMVRGA 0.100 175 RCRRLRGWWP 0.100 276FVNPLKTFVF 0.100 61 KGLEHDKQET 0.100 224 DMGGNVYILT 0.100 178 RLRGWWPVVK0.100 194 VEREAQEAQA 0.100 114 AEFRQPAVLV 0.090 Table XVI: 158P3D2 v.2aB7-10mers 110 IPQNRPIKLL 120.000 164 NPIFGEILEL 80.000 3 DPGDSDGVNL80.000 121 RVYVVKATNL 20.000 82 LVGKFKGSFL 20.000 38 ATLKIYNRSL 12.000119 LVRVYVVKAT 5.000 91 LIYPESEAVL 4.000 197 DLIGETHIDL 4.000 211YSHHRANCGL 4.000 31 VSPKKAVATL 4.000 162 QLNPIFGEIL 4.000 174 SISLPAETEL4.000 134 DPNGKADPYV 4.000 109 GIPQNRPIKL 4.000 183 LTVAVFEHDL 4.000 177LPAETELTVA 2.000 73 GGEEEGSGHL 1.200 97 EAVLFSEPQI 1.200 184 TVAVFEHDLV1.000 207 ENRFYSHHRA 1.000 131 APADPNGKAD 0.600 124 VVKATNLAPA 0.500 30TVSPKKAVAT 0.500 130 LAPADPNGKA 0.450 83 VGKFKGSFLI 0.400 161 KQLNPIFGEI0.400 10 VNLISMVGEI 0.400 114 RPIKLLVRVY 0.400 168 GEILELSISL 0.400 49EEFNHFEDWL 0.400 98 AVLFSEPQIS 0.300 147 AGRERQDTKE 0.300 136 NGKADPYVVV0.300 6 DSDGVNLISM 0.300 186 AVFEHDLVGS 0.300 28 KGTVSPKKAV 0.300 32SPKKAVATLK 0.200 219 GLASQYEVWV 0.200 61 FPLYRGQGGQ 0.200 18 EIQDQGEAEV0.200 217 NCGLASQYEV 0.200 103 EPQISRGIPQ 0.200 51 FNHFEDWLNV 0.200 140DPYVVVSAGR 0.200 90 FLIYPESEAV 0.200 159 IPKQLNPIFG 0.200 176 SLPAETELTV0.200 43 YNRSLEEEFN 0.200 106 ISRGIPQNRP 0.150 36 AVATLKIYNR 0.150 145VSAGRERQDT 0.150 227 WVQQGPQEPF 0.150 93 YPESEAVLFS 0.120 154 TKERYIPKQL0.120 189 EHDLVGSDDL 0.120 54 FEDWLNVFPL 0.120 101 FSEPQISRGI 0.120 29GTVSPKKAVA 0.100 112 QNRPIKLLVR 0.100 175 ISLPAETELT 0.100 21 DQGEAEVKGT0.100 193 VGSDDLIGET 0.100 118 LLVRVYVVKA 0.100 172 ELSISLPAET 0.100 127ATNLAPADPN 0.090 138 KADPYVVVSA 0.090 143 VVVSAGRERQ 0.075 59 NVFPLYRGQG0.075 35 KAVATLKIYN 0.060 215 RANCGLASQY 0.060 37 VATLKIYNRS 0.060 22QGEAEVKGTV 0.060 192 LVGSDDLIGE 0.050 142 YVVVSAGRER 0.050 144VVSAGRERQD 0.050 9 GVNLISMVGE 0.050 225 EVWVQQGPQE 0.050 123 YVVKATNLAP0.050 26 EVKGTVSPKK 0.050 15 MVGEIQDQGE 0.050 107 SRGIPQNRPI 0.040 166IFGEILELSI 0.040 157 RYIPKQLNPI 0.040 190 HDLVGSDDLI 0.040 150ERQDTKERYI 0.040 220 LASQYEVWVQ 0.030 216 ANCGLASQYE 0.030 126KATNLAPADP 0.030 146 SAGRERQDTK 0.030 13 ISMVGEIQDQ 0.030 221 ASQYEVWVQQ0.030 185 VAVFEHDLVG 0.030 115 PIKLLVRVYV 0.030 155 KERYIPKQLN 0.030 16VGEIQDQGEA 0.030 170 ILELSISLPA 0.030 158 YIPKQLNPIF 0.020 7 SDGVNLISMV0.020 149 RERQDTKERY 0.020 Table XVI: 158P3D2 v.3 B7-10mers 8 SVRRRSGPFA5.000 9 VRRRSGPFAL 4.000 1 LPTEREVSVR 0.200 6 EVSVRRRSGP 0.075 7VSVRRRSGPF 0.020 10 RRRSGPFALE 0.015 3 TEREVSVRRR 0.010 5 REVSVRRRSG0.002 4 EREVSVRRRS 0.001 2 PTEREVSVRR 0.000 Table XVI: 158P3D2 v.4B7-10mers 10 IWRRSGPFAL 4.000 1 YLPTEREVSI 0.400 2 LPTEREVSIW 0.400 9SIWRRSGPFA 0.100 7 EVSIWRRSGP 0.075 8 VSIWRRSGPF 0.020 4 TEREVSIWRR0.010 6 REVSIWRRSG 0.002 5 EREVSIWRRS 0.001 3 PTEREVSIWR 0.000 TableXVI: 158P3D2 v.5a B7-10mers 9 YTASLPMTSL 6.000 30 VGPGAPSSAL 6.000 4LQVWDYTASL 4.000 42 WPAMGPGRGA 3.000 46 GPGRGAICFA 2.000 31 GPGAPSSALC2.000 13 LPMTSLDPWS 1.200 36 SSALCSWPAM 1.000 34 APSSALCSWP 0.600 43PAMGPGRGAI 0.540 29 CVGPGAPSSA 0.500 2 LVLQVWDYTA 0.500 19 DPWSCSYQTW0.400 44 AMGPGRGAIC 0.300 21 WSCSYQTWCV 0.200 1 VLVLQVWDYT 0.100 25YQTWCVGPGA 0.100 49 RGAICFAAAA 0.100 47 PGRGAICFAA 0.100 33 GAPSSALCSW0.060 5 QVWDYTASLP 0.050 38 ALCSWPAMGP 0.045 15 MTSLDPWSCS 0.030 11ASLPMTSLDP 0.030 10 TASLPMTSLD 0.030 37 SALCSWPAMG 0.030 6 VWDYTASLPM0.030 45 MGPGRGAICF 0.020 16 TSLDPWSCSY 0.020 12 SLPMTSLDPW 0.020 28WCVGPGAPSS 0.020 3 VLQVWDYTAS 0.020 48 GRGAICFAAA 0.010 22 SCSYQTWCVG0.010 23 CSYQTWCVGP 0.010 18 LDPWSCSYQT 0.010 14 PMTSLDPWSC 0.010 7WDYTASLPMT 0.010 35 PSSALCSWPA 0.010 39 LCSWPAMGPG 0.010 26 QTWCVGPGAP0.010 40 CSWPAMGPGR 0.010 27 TWCVGPGAPS 0.003 17 SLDPWSCSYQ 0.003 8DYTASLPMTS 0.002 32 PGAPSSALCS 0.002 24 SYQTWCVGPG 0.001 41 SWPAMGPGRG0.001 20 PWSCSYQTWC 0.001

[0775] TABLE XVII 158P3D2 B35, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ.ID Pos 123456789 Score NO. Table XVII: 158P3D2 v.1 B35-9mers 268RPKTSFNWF 120.000 265 KPSRPKTSF 40.000 278 NPLKTFVFF 20.000 214KGRPEDLEF 9.000 314 IPGQISQVI 8.000 94 LPTEREVSV 8.000 10 VPAPPPVDI8.000 154 GPELCSVQL 6.000 168 GPRCNLFRC 6.000 255 KGRKQPEPL 6.000 133SANDFLGSL 6.000 318 ISQVIFRPL 5.000 75 NSLTGEGNF 5.000 118 QPAVLVLQV4.000 24 ISYELRVVI 4.000 22 QPISYELRV 4.000 38 VVLDDENPL 3.000 55YVKSWVKGL 3.000 175 RCRRLRGWW 3.000 166 GAGPRCNLF 3.000 180 RGWWPVVKL2.000 183 WPVVKLKEA 2.000 283 FVFFIWRRY 2.000 304 TVFLLLVFY 2.000 121VLVLQVWDY 2.000 44 NPLTGEMSS 2.000 19 KPRQPISYE 1.200 178 RLRGWWPVV1.200 299 LLVLLTVFL 1.000 165 NGAGPRCNL 1.000 224 DMGGNVYIL 1.000 277VNPLKTFVF 1.000 298 LLLVLLTVF 1.000 294 TLVLLLLVL 1.000 137 FLGSLELQL1.000 171 CNLFRCRRL 1.000 101 SVWRRSGPF 1.000 81 GNFNWRFVF 1.000 300LVLLTVFLL 1.000 50 MSSDIYVKS 1.000 83 FNWRFVFRF 1.000 232 LTGKVEAEF1.000 303 LTVFLLLVF 1.000 301 VLLTVFLLL 1.000 295 LVLLLLVLL 1.000 77LTGEGNFNW 1.000 235 KVEAEFELL 0.900 151 GARGPELCS 0.900 46 LTGEMSSDI0.800 51 SSDIYVKSW 0.750 132 ISANDFLGS 0.750 222 FTDMGGNVY 0.600 47TGEMSSDIY 0.600 259 QPEPLEKPS 0.600 140 SLELQLPDM 0.600 212 RRKGRPEDL0.600 124 LQVWDYDRI 0.600 293 RTLVLLLLV 0.400 306 FLLLVFYTI 0.400 251RPVGKGRKQ 0.400 6 FPQDVPAPP 0.400 261 EPLEKPSRP 0.400 129 YDRISANDF0.300 291 YWRTLVLLL 0.300 17 DIKPRQPIS 0.300 27 ELRVVIWNT 0.300 287IWRRYWRTL 0.300 69 ETDVHFNSL 0.300 103 WRRSGPFAL 0.300 237 EAEFELLTV0.270 216 RPEDLEFTD 0.240 164 RNGAGPRCN 0.200 234 GKVEAEFEL 0.200 30VVIWNTEDV 0.200 313 TIPGQISQV 0.200 18 IKPRQPISY 0.200 150 RGARGPELC0.200 297 LLLLVLLTV 0.200 42 DENPLTGEM 0.200 107 GPFALEEAE 0.200 290RYWRTLVLL 0.200 302 LLTVFLLLV 0.200 12 APPPVDIKP 0.200 31 VIWNTEDVV0.200 276 FVNPLKTFV 0.200 228 NVYILTGKV 0.200 125 QVWDYDRIS 0.200 86RFVFRFDYL 0.200 144 QLPDMVRGA 0.200 66 DKQETDVHF 0.200 80 EGNFNWRFV0.200 85 WRFVFRFDY 0.200 289 RRYWRTLVL 0.200 270 KTSFNWFVN 0.200 113EAEFRQPAV 0.180 190 EAEDVEREA 0.180 76 SLTGEGNFN 0.150 266 PSRPKTSFN0.150 32 IWNTEDVVL 0.150 119 PAVLVLQVW 0.150 Table XVII: 158P3D2 v.2aB35-9-mers 32 SPKKAVATL 60.000 159 IPKQLNPIF 60.000 134 DPNGKADPY 40.000110 IPQNRPIKL 20.000 35 KAVATLKIY 12.000 93 YPESEAVLF 9.000 177LPAETELTV 8.000 114 RPIKLLVRV 8.000 78 GSGHLVGKF 5.000 175 ISLPAETEL5.000 131 APADPNGKA 4.000 3 DPGDSDGVN 4.000 39 TLKIYNRSL 3.000 83VGKFKGSFL 3.000 43 YNRSLEEEF 3.000 198 LIGETHIDL 2.000 216 ANCGLASQY2.000 169 EILELSISL 2.000 85 KFKGSFLIY 1.200 228 VQQGPQEPF 1.000 184TVAVFEHDL 1.000 82 LVGKFKGSF 1.000 163 LNPIFGEIL 1.000 136 NGKADPYVV0.900 203 HIDLENRFY 0.900 185 VAVFEHDLV 0.900 46 SLEEEFNHF 0.900 108RGIPQNRPI 0.800 155 KERYIPKQL 0.600 112 QNRPIKLLV 0.600 220 LASQYEVWV0.600 115 PIKLLVRVY 0.600 88 GSFLIYPES 0.500 219 GLASQYEVW 0.500 173LSISLPAET 0.500 31 VSPKKAVAT 0.500 146 SAGRERQDT 0.450 158 YIPKQLNPI0.400 11 NLISMVGEI 0.400 191 DLVGSDDLI 0.400 98 AVLFSEPQI 0.400 117KLLVRVYVV 0.400 162 QLNPIFGEI 0.400 150 ERQDTKERY 0.400 194 GSDDLIGET0.300 91 LIYPESEAV 0.300 119 LVRVYVVKA 0.300 45 RSLEEEFNH 0.300 180ETELTVAVF 0.300 151 RQDTKERYI 0.240 140 DPYVVVSAG 0.200 121 RVYVVKATN0.200 202 THIDLENRF 0.200 28 KGTVSPKKA 0.200 7 SDGVNLISM 0.200 56DWLNVFPLY 0.200 8 DGVNLISMV 0.200 164 NPIFGEILE 0.200 218 CGLASQYEV0.200 29 GTVSPKKAV 0.200 61 FPLYRGQGG 0.200 92 IYPESEAVL 0.200 103EPQISRGIP 0.200 138 KADPYVVVS 0.180 165 PIFGEILEL 0.150 106 ISRGIPQNR0.150 176 SLPAETELT 0.150 6 DSDGVNLIS 0.150 51 FNHFEDWLN 0.150 99VLFSEPQIS 0.150 71 DGGGEEEGS 0.150 167 FGEILELSI 0.120 212 SHHRANCGL0.100 50 EFNHFEDWL 0.100 36 AVATLKIYN 0.100 90 FLIYPESEA 0.100 81HLVGKFKGS 0.100 122 VYVVKATNL 0.100 105 QISRGIPQN 0.100 190 HDLVGSDDL0.100 38 ATLKIYNRS 0.100 123 YVVKATNLA 0.100 111 PQNRPIKLL 0.100 128TNLAPADPN 0.100 55 EDWLNVFPL 0.100 30 TVSPKKAVA 0.100 24 EAEVKGTVS 0.09034 KKAVATLKI 0.080 5 GDSDGVNLI 0.080 221 ASQYEVWVQ 0.075 52 NHFEDWLNV0.060 126 KATNLAPAD 0.060 215 RANCGLASQ 0.060 153 DTKERYIPK 0.060 147AGRERQDTK 0.060 53 HFEDWLNVF 0.060 19 IQDQGEAEV 0.060 74 GEEEGSGHL 0.06049 EEFNHFEDW 0.050 145 VSAGRERQD 0.050 Table XVII: 158P3D2 v.3B35-9-mers 7 SVRRRSGPF 3.000 9 RRRSGPFAL 0.600 6 VSVRRRSGP 0.050 8VRRRSGPFA 0.030 4 REVSVRRRS 0.020 5 EVSVRRRSG 0.010 2 TEREVSVRR 0.006 1PTEREVSVR 0.000 3 EREVSVRRR 0.000 Table XVII: 158P3D2 v.4 B35-9-mers 1LPTEREVSI 16.000 8 SIWRRSGPF 1.000 7 VSIWRRSGP 0.050 9 IWRRSGPFA 0.030 2PTEREVSIW 0.022 5 REVSIWRRS 0.020 6 EVSIWRRSG 0.010 3 TEREVSIWR 0.006 4EREVSIWRR 0.000 Table XVII: 158P3D2 v.5a B35-9-mers 45 GPGRGAICF 20.00030 GPGAPSSAL 20.000 33 APSSALCSW 10.000 12 LPMTSLDPW 10.000 36 SALCSWPAM6.000 9 TASLPMTSL 3.000 4 QVWDYTASL 2.000 18 DPWSCSYQT 2.000 15TSLDPWSCS 1.000 16 SLDPWSCSY 0.600 20 WSCSYQTWC 0.500 35 SSALCSWPA 0.50043 AMGPGRGAI 0.400 49 GAICFAAAA 0.300 32 GAPSSALCS 0.300 6 WDYTASLPM0.200 41 WPAMGPGRG 0.200 21 SCSYQTWCV 0.200 48 RGAICFAAA 0.200 3LQVWDYTAS 0.150 14 MTSLDPWSC 0.150 1 LVLQVWDYT 0.100 2 VLQVWDYTA 0.10027 WCVGPGAPS 0.100 25 QTWCVGPGA 0.100 29 VGPGAPSSA 0.100 44 MGPGRGAIC0.100 28 CVGPGAPSS 0.100 8 YTASLPMTS 0.100 10 ASLPMTSLD 0.050 22CSYQTWCVG 0.050 39 CSWPAMGPG 0.050 42 PAMGPGRGA 0.030 46 PGRGAICFA 0.03013 PMTSLDPWS 0.010 37 ALCSWPAMG 0.010 38 LCSWPAMGP 0.010 7 DYTASLPMT0.010 11 SLPMTSLDP 0.010 24 YQTWCVGPG 0.010 31 PGAPSSALC 0.010 47GRGAICFAA 0.010 34 PSSALCSWP 0.005 19 PWSCSYQTW 0.005 17 LDPWSCSYQ 0.00126 TWCVGPGAP 0.001 23 SYQTWCVGP 0.001 40 SWPAMGPGR 0.001 5 VWDYTASLP0.000

[0776] TABLE XVIII 158P3D2 B35, 10mers (variants 1, 2a, 3, 4 and 5a)SEQ. ID Pos 1234567890 Score NO. Table XVIII: 158P3D2 v.1 B35-10mers 19KPRQPISYEL 120.000 216 RPEDLEFTDM 72.000 94 LPTEREVSVW 30.000 107GPFALEEAEF 30.000 268 RPKTSFNWFV 24.000 139 GSLELQLPDM 20.000 314IPGQISQVIF 20.000 118 QPAVLVLQVW 10.000 278 NPLKTFVFFI 8.000 17DIKPRQPISY 6.000 22 QPISYELRVV 6.000 271 TSFNWFVNPL 5.000 24 ISYELRVVIW5.000 50 MSSDIYVKSW 5.000 132 ISANDFLGSL 5.000 100 VSVWRRSGPF 5.000 46LTGEMSSDIY 4.000 153 RGPELCSVQL 4.000 265 KPSRPKTSFN 4.000 148MVRGARGPEL 3.000 233 TGKVEAEFEL 3.000 151 GARGPELCSV 2.700 164RNGAGPRCNL 2.000 120 AVLVLQVWDY 2.000 293 RTLVLLLLVL 2.000 303LTVFLLLVFY 2.000 170 RCNLFRCRRL 2.000 37 DVVLDDENPL 1.500 31 VIWNTEDVVL1.500 298 LLLVLLTVFL 1.000 317 QISQVIFRPL 1.000 294 TLVLLLLVLL 1.000 286FIWRRYWRTL 1.000 299 LLVLLTVFLL 1.000 300 LVLLTVFLLL 1.000 277VNPLKTFVFF 1.000 105 RSGPFALEEA 1.000 302 LLTVFLLLVF 1.000 74 FNSLTGEGNF1.000 231 ILTGKVEAEF 1.000 80 EGNFNWRFVF 1.000 297 LLLLVLLTVF 1.000 165NGAGPRCNLF 1.000 276 FVNPLKTFVF 1.000 113 EAEFRQPAVL 0.900 185VVKLKEAEDV 0.900 214 KGRPEDLEFT 0.900 162 LARNGAGPRC 0.900 75 NSLTGEGNFN0.750 266 PSRPKTSFNW 0.750 123 VLQVWDYDRI 0.600 154 GPELCSVQLA 0.600 84NWRFVFRFDY 0.600 211 RRRKGRPEDL 0.600 61 KGLEHDKQET 0.600 168 GPRCNLFRCR0.600 158 CSVQLARNGA 0.500 283 FVFFIWRRYW 0.500 76 SLTGEGNFNW 0.500 9DVPAPPPVDI 0.400 261 EPLEKPSRPK 0.400 29 RVVIWNTEDV 0.400 21 RQPISYELRV0.400 251 RPVGKGRKQP 0.400 309 LVFYTIPGQI 0.400 6 FPQDVPAPPP 0.400 258KQPEPLEKPS 0.400 117 RQPAVLVLQV 0.400 313 TIPGQISQVI 0.400 221EFTDMGGNVY 0.400 213 RKGRPEDLEF 0.300 125 QVWDYDRISA 0.300 129YDRISANDFL 0.300 102 VWRRSGPFAL 0.300 115 EFRQPAVLVL 0.300 288WRRYWRTLVL 0.300 134 ANDFLGSLEL 0.300 78 TGEGNFNWRF 0.300 38 VVLDDENPLT0.300 234 GKVEAEFELL 0.300 65 HDKQETDVHF 0.300 291 YWRTLVLLLL 0.300 12APPPVDIKPR 0.300 131 RISANDFLGS 0.300 44 NPLTGEMSSD 0.300 51 SSDIYVKSWV0.300 290 RYWRTLVLLL 0.200 282 TFVFFIWRRY 0.200 183 WPVVKLKEAE 0.200 10VPAPPPVDIK 0.200 68 QETDVHFNSL 0.200 227 GNVYILTGKV 0.200 93 YLPTEREVSV0.200 30 VVIWNTEDVV 0.200 296 VLLLLVLLTV 0.200 150 RGARGPELCS 0.200 301VLLTVFLLLV 0.200 289 RRYWRTLVLL 0.200 312 YTIPGQISQV 0.200 187KLKEAEDVER 0.180 Table XVIII: 158P3D2 v.2a B35-10mers 114 RPIKLLVRVY80.000 3 DPGDSDGVNL 60.000 45 RSLEEEFNHF 30.000 164 NPIFGEILEL 30.000110 IPQNRPIKLL 20.000 215 RANCGLASQY 12.000 177 LPAETELTVA 6.000 211YSHHRANCGL 5.000 31 VSPKKAVATL 5.000 134 DPNGKADPYV 4.000 6 DSDGVNLISM3.000 121 RVYVVKATNL 2.000 83 VGKFKGSFLI 1.200 97 EAVLFSEPQI 1.200 149RERQDTKERY 1.200 201 ETHIDLENRF 1.000 91 LIYPESEAVL 1.000 81 HLVGKFKGSF1.000 158 YIPKQLNPIF 1.000 77 EGSGHLVGKF 1.000 227 WVQQGPQEPF 1.000 109GIPQNRPIKL 1.000 162 QLNPIFGEIL 1.000 197 DLIGETHIDL 1.000 183LTVAVFEHDL 1.000 82 LVGKFKGSFL 1.000 174 SISLPAETEL 1.000 38 ATLKIYNRSL1.000 161 KQLNPIFGEI 0.800 145 VSAGRERQDT 0.750 175 ISLPAETELT 0.750 32SPKKAVATLK 0.600 93 YPESEAVLFS 0.600 202 THIDLENRFY 0.600 159 IPKQLNPIFG0.600 35 KAVATLKIYN 0.600 73 GGEEEGSGHL 0.600 101 FSEPQISRGI 0.600 136NGKADPYVVV 0.600 218 CGLASQYEVW 0.500 43 YNRSLEEEFN 0.450 131 APADPNGKAD0.400 18 EIQDQGEAEV 0.400 28 KGTVSPKKAV 0.400 10 VNLISMVGEI 0.400 34KKAVATLKIY 0.400 92 IYPESEAVLF 0.300 130 LAPADPNGKA 0.300 37 VATLKIYNRS0.300 186 AVFEHDLVGS 0.300 124 VVKATNLAPA 0.300 51 FNHFEDWLNV 0.300 90FLIYPESEAV 0.300 184 TVAVFEHDLV 0.300 207 ENRFYSHHRA 0.300 21 DQGEAEVKGT0.300 119 LVRVYVVKAT 0.300 179 AETELTVAVF 0.200 61 FPLYRGQGGQ 0.200 176SLPAETELTV 0.200 193 VGSDDLIGET 0.200 217 NCGLASQYEV 0.200 52 NHFEDWLNVF0.200 133 ADPNGKADPY 0.200 219 GLASQYEVWV 0.200 140 DPYVVVSAGR 0.200 87KGSFLIYPES 0.200 55 EDWLNVFPLY 0.200 84 GKFKGSFLIY 0.200 103 EPQISRGIPQ0.200 138 KADPYVVVSA 0.180 106 ISRGIPQNRP 0.150 98 AVLFSEPQIS 0.150 29GTVSPKKAVA 0.100 49 EEFNHFEDWL 0.100 118 LLVRVYVVKA 0.100 127 ATNLAPADPN0.100 172 ELSISLPAET 0.100 42 IYNRSLEEEF 0.100 30 TVSPKKAVAT 0.100 168GEILELSISL 0.100 166 IFGEILELSI 0.080 157 RYIPKQLNPI 0.080 150ERQDTKERYI 0.080 13 ISMVGEIQDQ 0.075 115 PIKLLVRVYV 0.060 155 KERYIPKQLN0.060 153 DTKERYIPKQ 0.060 126 KATNLAPADP 0.060 147 AGRERQDTKE 0.060 22QGEAEVKGTV 0.060 173 LSISLPAETE 0.050 78 GSGHLVGKFK 0.050 88 GSFLIYPESE0.050 221 ASQYEVWVQQ 0.050 220 LASQYEVWVQ 0.045 167 FGEILELSIS 0.045 16VGEIQDQGEA 0.045 107 SRGIPQNRPI 0.040 190 HDLVGSDDLI 0.040 Table XVIII:158P3D2 v.3 B35-10mers 7 VSVRRRSGPF 5.000 1 LPTEREVSVR 0.600 8SVRRRSGPFA 0.300 9 VRRRSGPFAL 0.300 6 EVSVRRRSGP 0.010 10 RRRSGPFALE0.006 3 TEREVSVRRR 0.006 4 EREVSVRRRS 0.003 5 REVSVRRRSG 0.002 2PTEREVSVRR 0.000 Table XVIII: 158P3D2 v.4 B35-10mers 2 LPTEREVSIW 30.0008 VSIWRRSGPF 5.000 1 YLPTEREVSI 0.400 10 IWRRSGPFAL 0.300 9 SIWRRSGPFA0.100 7 EVSIWRRSGP 0.010 4 TEREVSIWRR 0.006 5 EREVSIWRRS 0.003 6REVSIWRRSG 0.002 3 PTEREVSIWR 0.000 Table XVIII: 158P3D2 v.5a B35-10mers16 TSLDPWSCSY 20.000 19 DPWSCSYQTW 10.000 36 SSALCSWPAM 10.000 31GPGAPSSALC 2.000 42 WPAMGPGRGA 2.000 46 GPGRGAICFA 2.000 13 LPMTSLDPWS2.000 33 GAPSSALCSW 1.500 30 VGPGAPSSAL 1.000 21 WSCSYQTWCV 1.000 9YTASLPMTSL 1.000 45 MGPGRGAICF 1.000 4 LQVWDYTASL 1.000 12 SLPMTSLDPW0.500 49 RGAICFAAAA 0.200 34 APSSALCSWP 0.200 3 VLQVWDYTAS 0.150 43PAMGPGRGAI 0.120 15 MTSLDPWSCS 0.100 1 VLVLQVWDYT 0.100 44 AMGPGRGAIC0.100 25 YQTWCVGPGA 0.100 2 LVLQVWDYTA 0.100 29 CVGPGAPSSA 0.100 28WCVGPGAPSS 0.100 6 VWDYTASLPM 0.060 40 CSWPAMGPGR 0.050 35 PSSALCSWPA0.050 11 ASLPMTSLDP 0.050 23 CSYQTWCVGP 0.050 47 PGRGAICFAA 0.030 37SALCSWPAMG 0.030 10 TASLPMTSLD 0.030 5 QVWDYTASLP 0.020 14 PMTSLDPWSC0.015 27 TWCVGPGAPS 0.010 26 QTWCVGPGAP 0.010 7 WDYTASLPMT 0.010 48GRGAICFAAA 0.010 22 SCSYQTWCVG 0.010 39 LCSWPAMGPG 0.010 38 ALCSWPAMGP0.010 18 LDPWSCSYQT 0.010 8 DYTASLPMTS 0.010 32 PGAPSSALCS 0.010 17SLDPWSCSYQ 0.003 24 SYQTWCVGPG 0.001 20 PWSCSYQTWC 0.001 41 SWPAMGPGRG0.001

[0777] TABLE XIXA MHC Class I Analysis of 158P3D2 (9-mers) Table XIXA,part 1: MHC Class I nonamer analysis of 158P3D2 v.1 (aa 1-328) Listedare scores which correlate with the ligation strength to a defined HLAtype for a sequence of amino acids. The algorithms used are based on thebook “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmannand S. Stevanovic. The probability of being processed and presented isgiven in order to predict T-cell epitopes Pos 1 2 3 4 5 6 7 8 9 scoreHLA-A*0201 nonamers 297 L L L L V L L T V 31 299 L L V L L T V F L 27302 L L T V F L L L V 27 294 T L V L L L L V L 26 133 S A N D F L G S L25 295 L V L L L L V L L 25 224 D M G G N V Y I L 24 301 V L L T V F L LL 24 306 F L L L V F Y T I .24 313 T I P G Q I S Q V 24 137 F L G S LE L Q L 23 178 R L R G W W P V V 23 296 V L L L L V L L T 23 230 Y I L TG K V E A 22 293 R T L V L L L L V 22 300 L V L L T V F L L 22 31 V I WN T E D V V 20 140 S L E L Q L P D M 20 144 Q L P D M V R G A 20 152 A RG P E L C S V 20 180 R G W W P V V K L 20 228 N V Y I L T G K V 20 2 W ID I F P Q D V 19 30 V V I W N T E D V 19 38 V V L D D E N P L 19 55 Y VK S W V K G L 19 231 I L T G K V E A E 19 272 S F N W F V N P L 19 276 FV N P L K T F V 19 279 P L K T F V F F I 19 298 L L L V L L T V F 19 23P I S Y E L R V V 18 116 P R Q P A V L V L 18 118 Q P A V L V L Q V 18291 Y W R T L V L L L 18 39 V L D D E N P L T 17 94 L P T E R E V S V 17290 R Y W R T L V L L 17 4 D I F P Q D V P A 16 10 V P A P P P V D I 1624 I S Y E L R V V I 16 46 L T G E M S S D I 16 62 G L E H D K Q E T 16135 N D F L G S L E L 16 237 E A E F E L L T V 16 27 E L R V V I W N T15 32 I W N T E D V V L 15 92 D Y L P T E R E V 15 114 A E F R Q P A V L15 121 V L V L Q V W D Y 15 141 L E L Q L P D M V 15 161 Q L A R N G A GP 15 165 N G A G P R C N L 15 223 T D M G G N V Y I 15 234 G K V E A E FE L 15 242 L L T V E E A E K 15 287 I W R R Y W R T L 15 307 L L L V FY T I P 15 HLA-A1 nonamers 222 F T D M G G N V Y 36 34 N T E D V V L D D25 47 T G E M S S D I Y 25 18 I K P R Q P I S Y 21 121 V L V L Q V W D Y20 69 E T D V F I F N S L 19 51 S S D I Y V K S W 18 95 P T E R E V S VW 18 312 Y T I P G Q I S Q 18 HLA-A26 nonamers 69 E T D V H F N S L 30304 T V F L L L V F Y 28 55 Y V K S W V K G L 25 303 L T V F L L L V F25 295 L V L L L L V L L 24 121 V L V L Q V W D Y 23 232 L T G K V E A EF 23 283 P V F F I W R R Y 23 298 L L L V L L T V F 23 4 D I F P Q D V PA 22 140 S L E L Q L P D M 22 235 K V E A E F E L L 22 300 L V L L T V FL L 22 222 F T D M G G N V Y 21 294 T L V L L L L V L 21 17 D I K P R QP I S 20 66 D K Q E T D V H F 20 101 S V W R R S G P F 20 224 D M G G NV Y I L 20 275 W F V N P L K T F 20 301 V L L T V F L L L 20 313 T I P GQ I S Q V 20 27 E L R V V I W N T 19 38 V V L D D E N P L 19 108 P F A LE E A E F 19 136 D F L G S L E L Q 19 137 F L G S L E L Q L 19 9 D V P AP P P V D 18 42 D E N P L T G E M 18 86 R F V F R F D Y L 18 193 D V E RE A Q E A 18 272 S F N W F V N P L 18 299 L L V L L T V F L 18 309 L V FY T I P G Q 18 37 D V V L D D E N P 17 53 D I Y V K S W V K 17 99 E V SV W R R S G 17 130 D R I S A N D F L 17 45 P L T G E M S S D 16 71 D V HF N S L T G 16 156 E L C S V Q L A R 16 219 D L E P T D M G G 16 231 I LT G K V E A E 16 268 R P K T S F N W F 16 278 N P L K T F V F F 16 281 KT F V F F I W R 16 317 Q I S Q V I F R P 16 34 N T E D V V L D D 15 83 FN W R F V F R F 15 95 P T E R E V S V W 15 142 E L Q L P D M V R 15 144Q L P D M V R G A 15 239 E F E L L T V E E 15 241 E L L T V E E A E 15286 F I W R R Y W R T 15 293 R T L V L L L L V 15 312 Y T I P G Q I S Q15 HLA-A3 nonamers 320 Q V I F R P L H K 31 53 D I Y V K S W V K 27 59 WV K G L E H D K 23 178 R L R G W W P V V 23 242 L L T V E E A E K 23 161Q L A R N G A G P 22 101 S V W R R S G P F 21 257 R K Q P E P L E K 21297 L L L L V L L T V 21 298 L L L V L L T V F 21 304 T V F L L L V F Y21 120 A V L V L Q VW D 20 142 E L Q L P D M V R 20 262 P L E K P S R PK 20 156 E L C S V Q L A R 19 179 L R G W W P V V K 19 187 K L K E AE D V E 19 198 A Q E A Q A G K K 19 172 N L F R C R R L R 18 294 T L V LL L L V L 18 306 F L L L V F Y T I 18 9 D V P A P P P V D 17 45 P L T GE M S S D 17 71 D V H F N S L T G 17 121 V L V L Q V W D Y 17 148 M VR G A R G P E 17 201 A Q A G K K K R K 17 206 K K R K Q R R R K 17 247 EA E K R P V G K 17 289 R R Y W R T L V L 17 295 L V L L L L V L L 17 15P V D I K P R Q P 16 24 I S Y E L R V V I 16 29 R V V I W N T E D 16 76S L T G E G N F N 16 137 F L G S L E L Q L 16 185 V V K L K E A E D 16193 D V E R E A Q E A 16 199 Q E A Q A G K K K 16 214 K G R P E D L E F16 228 N V Y I L T G K V 16 230 Y I L T G K V E A 16 231 I L T G K V E AE 16 250 K R P V G K G R K 16 252 P V G K G R K Q P 16 283 F V F F IW R R Y 16 296 V L L L L V L L T 16 301 V L L T V F L L L 16 313 T I P GQ I S Q V 16 4 D I F P Q D Y P A 15 93 Y L P T E R E V S 15 222 F T D MG G N V Y 15 235 K V E A E F E L L 15 299 L L V L L T V F L 15HLA-B*0702 nonamers 10 V P A P P P V D I 23 265 K P S R P K T S F 23 154G P E L C S V Q L 22 278 N P L K T F V F F 21 118 Q P A V L V L Q V 20314 I P G Q I S Q V I 19 22 Q P I S Y E L R V 18 94 L P T E R E V S V 18268 R P K T S F N W F 18 165 N G A G P R C N L 17 180 R G W W P V V K L17 19 K P R Q P I S Y E 16 183 W P V V K L K E A 16 116 F R Q P A V L VL 15 255 K G R K Q P E P L 15 289 R R Y W R T L V L 15 291 Y W R T L V LL L 15 32 I W N T E D V V L 14 114 A E F R Q P A V L 14 115 E F R Q P AV L V 14 149 V R G A R G P E L 14 224 D M G G N V Y I L 14 251 R P V G KG R K Q 14 299 L L V L L T V F L 14 12 A P P P V D I K P 13 69 E T D V HF N S L 13 103 W R R S G P F A L 13 137 F L G S L E L Q L 13 145 L P D MV R G A R 13 178 R L R G W W P V V 13 212 R R K G R P E D L 13 235 K V EA E F E L L 13 287 I W R R Y W R T L 13 290 R Y W R T L V L L 13 294 T LV L L L L V L 13 301 V L L T V F L L L 13 318 I S Q V I F R P L 13 6 F PO D V P A P P 12 16 V D I K P R Q P I 12 86 R F V F R F D Y L 12 107 G PF A L E E A E 12 135 N D F L G S L E L 12 168 G P R C N L F R C 12 214 KG R P E D L E F 12 259 Q P E P L E K P S 12 272 S F N W F V N P L 12 292W R T L V L L L L 12 295 L V L L L L V L L 12 13 P P P V D I K P R 11 14P P V D I K P R Q 11 20 P R Q P I S Y E L 11 24 I S Y E L R V V I 11 38V V L D D E N P L 11 55 Y V K S W V K G L 11 88 V F R F D Y L P T 11 102V W R R S G P F A 11 130 D R I S A N D F L 11 216 R P E D L E F T D 11223 T D M G G N V Y I 11 261 E P L E K P S R P 11 300 L V L L T V F L L11 HLA-B*08 nonamers 212 R R K G R P E D L 28 185 V V K L K E A E D 23279 P L K T F V F F I 23 17 D I K P R Q P I S 22 55 Y V K S W V K G L 22268 R P K T S F N W F 22 203 A G K K K R K Q R 21 149 V R G A R G P E L20 205 K K K R K Q R R R 20 261 E P L E K P S R P 20 154 G P E L C S V QL 19 166 G A G P R C N L F 19 183 W P V V K L K E A 19 204 G K K K R K QR R 19 231 I L T G K V E A E 19 253 V G K G R K Q P E 19 86 R F V F R FD Y L 18 171 C N L F R C R R L 18 187 K L K E A E D V E 18 207 K R K Q RR R K G 18 277 V N P L K T F V F 18 289 R R Y W R T L V L 18 299 L L V LL T V F L 18 94 L P T E R E V S V 17 103 W R R S G P F A L 17 137 F L GS L E L Q L 17 287 I W R R Y W R T L 17 291 Y W R T L V L L L 17 294 T LV L L L L V L 17 301 V L L T V F L L L 17 27 E L R V V I W N T 16 101 SV W R R S G P F 16 133 S A N D F L G S L 16 210 Q R R R K G R P E 16 251R P V G K G R K Q 16 255 K G R K Q P E P L 16 266 P S R P K T S F N 1653 D I Y V K S W V K 15 113 E A E F R Q P A V 15 176 C R R L R G W W P15 247 E A E K R P V G K 15 10 V P A P P P V D I 14 173 L F R C R R L RG 14 202 Q A G K K K R K Q 14 209 K Q R R R K G R P 14 234 G K V E A E FE L 14 246 E E A E K R P V G 14 306 F L L L V F Y T I 14 HLA-B*1510nonamers 32 I W N T E D V V L 16 116 F R Q P A V L V L 15 287 I W R R YW R T L 15 318 I S Q V I F R P L 15 154 G P E L G S V Q L 14 165 N G A GP R C N L 14 171 C N L F R C R R L 14 180 R G W W P V V K L 14 20 P R QP I S Y E L 13 103 W R R S G P F A L 13 114 A E F R Q P A V L 13 224 D MG G N V Y I L 13 234 G K V E A E F E L 13 294 T L V L L L L V L 13 55 YV K S W V K G L 12 64 E H D K Q E T D V 12 69 E T D V H F N S L 12 135 ND F L G S L E L 12 149 V R G A R G P E L 12 212 R R K G R P E D L 12 255K G R K Q P E P L 12 289 R R Y W R T L V L 12 290 R Y W R T L V L L 12291 Y W R T L V L L L 12 295 L V L L L L V L L 12 299 L L V L L T V F L12 38 V V L D D E N P L 11 133 S A N D F L G S L 11 235 K V E A E F E LL 11 272 S F N W F V N P L 11 300 L V L L T V F L L 11 72 V H F N S L TG E 10 81 G N F N W R F V F 10 86 R F V F R F D Y L 10 130 D R I S A N DF L 10 137 F L G S L E L Q L 10 292 W R T L V L L L L 10 301 V L L T V FL L L 10 42 D E N P L T G E M 9 66 D K Q E T D V H F 9 79 G E G N F N WR F 9 83 F N W R F V F R F 9 166 G A G P R C N L F 9 214 K G R P E D L EF 9 265 K P S R P K T S F 9 278 N P L K T F V F F 9 298 L L L V L L T VF 9 24 I S Y E L R V V I 8 108 P F A L E E A E F 8 140 S L E L Q L P D M8 232 L T G K V E A E F 8 275 W F V N P L K T F 8 277 V N P L K T F V F8 303 L T V F L L L V F 8 315 P G Q I S Q V I F 8 HLA-B*2705 nonamers289 R R Y W R T L V L 28 250 K R P V G K G R K 27 212 R R K G R P E D L26 20 P R Q P I S Y E L 25 97 E R E V S V W R R 25 116 F R Q P A V L V L24 292 W R T L V L L L L 24 130 D R I S A N D P L 23 103 W R R S G P F AL 22 149 V R G A R G P E L 22 179 L R G W W P V V K 22 85 W R F V F R FD Y 21 169 P R C N L F R C R 21 211 R R R K G R P E D 20 135 N D P L G SL E L 19 177 R R L R G W W P V 19 180 R G W W P V V K L 19 90 R F D Y LP T E R 18 104 R R S G P F A L E 18 204 G K K K R K Q R R 18 227 G N V YI L T G K 18 257 R K Q P E P L E K 18 79 G E G N F N W R F 17 81 G N F NW R F V F 17 154 G P E L C S V Q L 17 170 R C N L F R C R R 17 201 A Q AG K K K R K 17 205 K K K R K Q R R R 17 214 K G R P E D L E F 17 256 G RK Q P E P L E 17 265 K P S R P K T S F 17 282 T F V F F I W R R 17 298 LL L V L L T V F 17 316 G Q I S Q V I F R 17 53 D I Y V K S W V K 16 75 NS L T G E G N F 16 89 F R F D Y L P T E 16 114 A E F R Q P A V L 16 163A R N G A G P R C 16 181 G W W P V V K L K 16 206 KK R K Q R R R K 16207 K R K Q R R R K G 16 234 G K V E A E F E L 16 243 L T V E E A E K R16 268 R P K T S F N W F 16 281 K T F V F F I W R 16 290 R Y W R T L V LL 16 294 T L V L L L L V L 16 295 L V L L L L V L L 16 21 R Q P I S Y EL R 15 32 I W N T E D V V L 15 49 E M S S D I Y V K 15 86 R F V F R F DY L 15 109 F A L E E A E F R 15 142 E L Q L P D M V R 15 152 A R G P E LC S V 15 165 N G A G P R C N L 15 166 G A G P R C N L F 15 200 E A Q A GK K K R 15 203 A G K K K R K Q R 15 215 G R P E D L E F T 15 232 L T G KV E A E F 15 260 P E P L E K P S R 15 267 S R P K T S F N W 15 278 N P LK T F V F F 15 303 L T V F L L L V F 15 304 T V F L L L V F Y 15 28 L RV V I W N T E 14 96 T E R E V S V W R 14 108 P F A L E E A E F 14 156 EL C S V Q L A R 14 171 C N L F R C R R L 14 188 L K E A E D V E R 14 197E A Q E A Q A G K 14 198 A Q E A Q A G K K 14 199 Q E A Q A G K K K 14208 R K Q R R R K G R 14 224 D M G G N V Y I L 14 255 K G R K Q P E P L14 262 P L E K P S R P K 14 275 W F V N P L K T F 14 283 F V F F I W R RY 14 299 L L V L L T V F L 14 300 L V L L T V F L L 14 301 V L L T V F LL L 14 315 P G Q I S Q V I F 14 HLA-B*2709 nonamers 289 R R Y W R T L VL 27 177 R R L R G W W P V 24 212 R R K G R P E D L 24 20 P R Q P I S YE L 23 116 F R Q P A V L V L 23 130 D R I S A N D F L 22 292 W R T L V LL L L 22 103 W R R S G P F A L 21 149 V R G A R G P E L 21 152 A R G P EL C S V 20 288 W R R Y W R T L V 18 180 R G W W P V V K L 16 86 R F V FR F D Y L 15 154 G P E L C S V Q L 15 211 R R R K G R P E D 15 290 R Y WR T L V L L 15 293 R T L V L L L L V 15 104 R R S G P F A L E 14 215 G RP E D L E F T 14 234 G K V E A E F E L 14 256 G R K Q P E P L E 14 38 VV L D D E N P L 13 81 G N F N W R F V F 13 89 F R F D Y L P T E 13 114 AE F R Q P A V L 13 135 N D F L G S L E L 13 137 F L G S L E L Q L 13 163A R N G A G P R C 13 171 C N L F R C R R L 13 178 R L R G W W P V V 13250 K R P V G K G R K 13 295 L V L L L L V L L 13 300 L V L L T V F L L13 301 V L L T V F L L L 13 HLA-B*4402 nonamers 114 A E F R Q P A V L 2879 G E G N F N W R F 21 191 A E D V E R E A Q 17 238 A E F E L L T V E17 116 F R Q P A V L V L 16 166 G A G P R C N L F 16 248 A E K R P V G KG 16 25 S Y E L R V V I W 15 51 S S D I Y V K S W 15 69 E T D V H F N SL 15 81 G N F N W R F V F 15 135 N D F L G S L E L 15 214 K G R P E D LE F 15 263 L E K P S R P K T 15 275 W F V N P L K T F 15 295 L V L L L LV L L 15 301 V L L T V F L L L 15 304 T V F L L L V F Y 15 18 I K P R QP I S Y 14 20 P R Q P I S Y E L 14 26 Y E L R V V I W N 14 42 D E N P LT G E M 14 174 F R C R R L R G W 14 246 E E A E K R P V G 14 277 V N P LK T F V F 14 278 N P L K T F V F F 14 283 F V F F I W R R Y 14 284 V F FI W R R Y W 14 289 R R Y W R T L V L 14 290 R Y W R T L V L L 14 291 Y WR T L V L L L 14 292 W R T L V L L L L 14 294 T L V L L L L V L 14 300 LV L L T V F L L 14 HLA-B*5101 nonamers 314 I P G Q I S Q V I 25 10 V P AP P P V D I 23 94 L P T E R E V S V 23 24 I S Y E L R V V I 22 237 E A EF E L L T V 22 22 Q P I S Y E L R V 21 118 Q P A V L V L Q V 21 113 E AE F R Q P A V 18 133 S A N D F L G S L 18 180 R G W W P V V K L 18 297 LL L L V L L T V 18 306 F L L L V F Y T I 18 154 G P E L C S V Q L 17 261E P L E K P S R P 17 278 N P L K T F V F F 17 310 V F Y T I P G Q I 1712 A P P P V D I K P 16 92 D Y L P T E R E V 16 109 F A L E E A E F R 16119 P A V L V L Q V W 16 6 F P Q D V P A P P 15 46 L T G E M S S D I 1580 E G N F N W R F V 15 202 Q A G K K K R K Q 15 228 N V Y I L T G K V15 251 R P V G K G R K Q 15 31 V I W N T E D V V 14 44 N P L T G E M S S14 145 L P D M V R G A R 14 165 N G A G P R C N L 14 190 E A E D V E R EA 14 200 E A Q A G K K K R 14 223 T D M G G N V Y I 14 255 K G R K Q P EP L 14 268 R P K T S F N W F 14 289 R R Y W R T L V L 14 301 V L L T V FL L L 14 11 P A P P P V D I K 13 13 P P P V D I K P R 13 23 P I S Y E LR V V 13 32 I W N T E D V V L 13 116 F R Q P A V L V L 13 124 L Q V W DY D R I 13 141 L E L Q L P D M V 13 162 L A R N G A G P R 13 183 W P V VK L K E A 13 186 V K L K E A E D V 13 216 R P E D L E F T D 13 224 D M GG N V Y I L 13 247 E A E K R P V G K 13 279 P L K T F V F F I 13 300 L VL L T V F L L 13 14 P P V D I K P R Q 12 16 V D I K P R Q P I 12 53 D IY V K S W V K 12 107 G P F A L E E A E 12 151 G A R G P E L C S 12 153 RG P E L C S V Q 12 168 G P R C N L F R C 12 178 R L R G W W P V V 12 197E A Q E A Q A G K 12 287 I W R R Y W R T L 12 291 Y W R T L V L L L 12293 R T L V L L L L V 12 294 T L V L L L L V L 12 295 L V L L L L V L L12 302 L L T V F L L L V 12 313 T I P G Q I S Q V 12 part 2: MHC Class Inonamer analysis of 158P3D2v.2a (aa1-236). Pos 1 2 3 4 5 6 7 8 9 scoreHLA-A*0201 nonamers 117 K L L V R V Y V V 28 11 N L I S M V G E I 26 165P I F G E I L E L 25 91 L I Y P E S E A V 24 158 Y I P K Q L N P I 23162 Q L N P I F G E I 23 39 T L K I Y N R S L 22 169 E I L E L S I S L22 198 L I G E T H I D L 22 32 S P K K A V A T L 19 90 F L I Y P E S E A19 114 R P I K L L V R V 19 119 L V R V Y V V K A 19 177 L P A E T E L TV 19 179 A E T E L T V A V 19 191 D L V G S D D L I 19 220 L A S Q Y E VW V 19 19 I Q D Q G E A E V 18 175 I S L P A E T E L 18 176 S L P A ET E L T 18 184 T V A V F E H D L 18 5 G D S D G V N L I 17 23 G E A E VK G T V 17 137 G K A D P Y V V V 17 14 S M V G E I Q D Q 16 29 G T V S PK K A V 16 34 KK A V A T L K I 16 92 I Y P E S E A V L 16 98 A V L F SE P Q I 16 116 I K L L V R V Y V 16 197 D L I G E T H I D 16 228 E A E FE L L T V 16 105 Q I S R G I P Q N 15 129 N L A P A D P N G 15 170 I L EL S I S L P 15 215 R A N C G L A S Q 15 218 C G L A S Q Y E V 15 8 D G VN L I S M V 14 46 S L E E E F N H F 14 52 N H F E D W L N V 14 74 G E EE G S G H L 14 110 I P Q N R P I K L 14 118 L L V R V Y V V K 14 138 K AD P Y V V V S 14 185 V A V F E H D L V 14 190 H D L V G S D D L 14HLA-A1 nonamers 203 H I D L E N R F Y 26 85 K F K G S F L I Y 25 6 D S DG V N L I S 24 56 D W L N V F P L Y 22 101 F S E P Q I S R G 21 180 ET E L T V A V F 18 115 P I K L L V R V Y 17 138 K A D P Y V V V S 17 167F G E I L E L S I 17 216 A N C G L A S Q Y 17 1 M D D P G D S D G 16 46S L E E E F N I H F 16 95 E S E A V L F S E 16 134 D P N G K A D P Y 1635 K A V A T L K I Y 15 132 P A D P N G K A D 15 150 E R Q D T K E R Y15 69 G Q D G G G E E E 14 75 E E E G S G H L V 14 93 Y P E S E A V L F14 148 G R E R Q D T K E 14 194 G S D D L I G E T 14 205 D L E N R F Y SH 14 16 V G E I Q D Q G E 13 154 T K E R Y I P K Q 13 170 I L E L S IS L P 13 189 E H D L V G S D D 13 195 S D D L I G E T H 13 HLA-A26nonamers 180 E T E L T V A V F 31 169 E I L E L S I S L 28 165 P I F G EI L E L 27 115 P I K L L V R V Y 26 46 S L E E E F N H F 25 26 E V K G TV S P K 24 85 K F K G S F L I Y 24 153 D T K E R Y I P K 23 82 L V G K FK G S F 22 53 H F E D W L N V F 21 56 D W L N V F P L Y 21 172 E L S I SL P A E 21 201 E T H I D L E N R 21 203 H I D L E N R F Y 21 50 E F N HF E D W L 20 59 N V F P L Y R G Q 20 198 L I G E T H I D L 20 18 E I Q DQ G E A E 19 134 D P N G K A D P Y 19 182 E L T V A V F E H 19 184 T V AV F E H D L 19 205 D L E N R F Y S H 19 39 T L K I Y N R S L 18 55 E D WL N V F P L 18 150 E R Q D T K E R Y 18 197 D L I G E T H I D 18 158 Y IP K Q L N P I 17 191 D L V G S D D L I 17 225 E V W V Q Q G P Q 17 11 NL I S M V G E I 16 38 A T L K I Y N R S 16 78 G S G H L V G K F 16 81 HL V G K F K G S 16 105 Q I S R G I P Q N 16 119 L V R V Y V V K A 16 32S P K K A V A T L 15 162 Q L N P I F G E I 15 183 L T V A V F E H D 15202 T H I D L E N R F 15 216 A N C G L A S Q Y 15 219 G L A S Q Y E V W15 HLA-A3 nonamers 118 L L V R V Y V V K 32 26 E V K G T V S P K 26 121R V Y V V K A T N 26 147 A G R E R Q D T K 23 20 Q D Q G E A E V K 21 30T V S P K K A V A 21 41 K I Y N R S L E E 21 117 K L L V R V Y V V 21186 A V F E H D L V G 21 62 P L Y R G Q G G Q 20 115 P I K L L V R V Y20 216 A N C G L A S Q Y 20 109 G I P Q N R P I K 19 205 D L E N R F Y SH 19 33 P K K A V A T L K 18 57 W L N V F P L Y R 18 98 A V L F S E P QI 18 105 Q I S R G I P Q N 18 82 L V G K F K G S F 17 119 L V R V YV V K A 17 124 V V K A T N L A P 17 143 V V V S A G R E R 17 174 S I S LP A E T E 17 9 G V N L I S M V G 16 46 S L E E E F N H F 16 77 E G S G HL V G K 16 79 S G H L V G K F K 16 90 F L I Y P E S E A 16 91 L I Y P ES E A V 16 162 Q L N P I F G E I 16 170 I L E L S I S L P 16 HLA-B*0702nonamers 32 S P K K A V A T L 23 131 A P A D P N G K A 22 110 I P Q N RP I K L 21 114 R P I K L L V R V 20 177 L P A E T E L T V 19 93 Y P E SE A V L F 18 159 I P K Q L N P I F 18 4 P G D S D G V N L 14 165 P I F GE I L E L 14 55 E D W L N V F P L 13 92 I Y P E S E A V L 13 111 P Q N RP I K L L 13 134 D P N G K A D P Y 13 137 G K A D P Y V V V 13 155 K E RY I P K Q L 13 175 I S L P A E T E L 13 3 D P G D S D G V N 12 83 V G KF K G S F L 12 103 E P Q I S R G I P 12 140 D P Y V V V S A G 12 179 A ET E L T V A V 12 228 V Q Q G P Q E P F 12 30 T V S P K K A V A 11 34 K KA V A T L K I 11 50 E F N H F E D W L 11 61 F P L Y R G Q G G 11 112 Q NR P I K L L V 11 119 L V R V Y V V K A 11 122 V Y V V K A T N L 11 139 AD P Y V V V S A 11 163 L N P I F G E I L 11 169 E I L E L S I S L 11 184T V A V F E H D L 11 198 L I G E T H I D L 11 HLA-B*08 nonamers 83 V G KF K G S F L 31 32 S P K K A V A T L 29 39 T L K I Y N R S L 27 110 I P QN R P I K L 25 159 I P K Q L N P I F 24 122 V Y V V K A T N L 22 153 D TK E R Y I P K 20 81 H L V G K F K G S 18 155 K E R Y I P K Q L 18 169 EI L E L S I S L 18 24 E A E V K G T V S 17 37 V A T L K I Y N R 17 46 SL E E E F N H F 16 61 F P L Y R G Q G G 16 115 P I K L L V R V Y 16 117K L L V R V Y V V i6 134 D P N G K A D P Y 16 147 A G R E R Q D T K 16151 R Q D T K E R Y I 16 165 P I F G E I L E L 16 198 L I G E T H I D L16 HLA-B*1510 nonamers 202 T H I D L E N R F 20 212 S HH R A N C G L 2092 I Y P E S E A V L 15 175 I S L P A E T E L 15 74 G E E E G S G H L 1480 G H L V G K F K G 14 32 S P K K A V A T L 13 39 T L K I Y N R S L 1355 E D W L N V F P L 13 110 I P Q N R P I K L 13 165 P I F G E I L E L13 184 T V A V F E H D L 13 4 P G D S D G V N L 12 111 P Q N R P I K L L12 169 E I L E L S I S L 12 190 H D L V G S D D L 12 50 E F N H F E D WL 11 52 N H F E D W L N V 11 122 V Y V V K A T N L 11 155 K E R Y I P KQ L 11 180 E T E L T V A V F 11 189 E H D L V G S D D 11 198 L I G E T HI D L 11 213 H H R A N C G L A 11 53 H F E D W L N V F 10 83 V G K F K GS F L 10 93 Y P E S E A V L F 10 159 I P K Q L N P I F 10 163 L N P I FG E I L 10 HLA-B*2705 nonamers 113 N R P I K L L V R 24 150 E R Q D T KE R Y 21 165 P I F G E I L E L 21 37 V A T L K I Y N R 18 45 R S L E E EF N H 18 148 G R E R Q D T K E 18 74 G E E E G S G H L 17 78 G S G H L VG K F 17 84 G K F K G S F L I 17 122 V Y V V K A T N L 17 149 R E R Q DT K E R 17 169 E I L E L S I S L 17 175 I S L P A E T E L 17 100 L F S EP Q I S R 16 106 I S R G I P Q N R 16 107 S R G I P Q N R P 16 109 G I PQ N R P I K 16 159 I P K Q L N P I F 16 202 T H I D L E N R F 16 208 N RF Y S H H R A 16 20 Q D Q G E A E V K 15 27 V K G T V S P K K 15 32 S PK K A V A T L 15 92 I Y P E S E A V L 15 147 A G R E R Q D T K 15 190 HD L V G S D D L 15 216 A N C G L A S Q Y 15 228 V Q Q G P Q E P F 15 26E V K G T V S P K 14 33 P K K A V A T L K 14 64 Y R G Q G G Q D G 14 73G G E E E G S G H 14 77 E G S G H L V G K 14 82 L V G K F K G S F 14 85K F K G S F L I Y 14 108 R G I P Q N R P I 14 110 I P Q N R P I K L 14111 P Q N R P I K L L 14 118 L L V R V Y V V K 14 141 P Y V V V S A G R14 155 K E R Y I P K Q L 14 156 E R Y I P K Q L N 14 180 E T E L T V A VF 14 4 R G D S D G V N L 13 5 G D S D G V N L I 13 46 S L E E E F N H F13 53 H F E D W L N V F 13 93 Y P E S E A V L F 13 114 R P I K L L V R V13 120 V R V Y V V K A T 13 157 R Y I P K Q L N P 13 201 E T H I D L E NR 13 35 K A V A T L K I Y 12 39 T L K I Y N R S L 12 43 Y N R S L E E EF 12 44 N R S L E E E F N 12 55 E D W L N V F P L 12 56 D W L N V F P LY 12 79 S G H L V G K F K 12 83 V G K F K G S F L 12 98 A V L F S E P QI 12 115 P I K L L V R V Y 12 134 D P N G K A D P Y 12 143 V V V S A G RE R 12 153 D T K E R Y I P K 12 196 D D L I G E T H I 12 198 L I G E T HI D L 12 HLA-B*2709 nonamers 114 R P I K L L V R V 15 4 P G D S D G V NL 14 108 R G I P Q N R P I 14 117 K L L V R V Y V V 14 155 K E R Y I P KQ L 14 175 I S L P A E T E L 14 29 G T V S P K K A V 13 52 N H F E D W LN V 13 74 G E E E G S G H L 13 84 G K F K G S F L I 13 98 A V L F S E PQ I 13 122 V Y V V K A T N L 13 148 G R E R Q D T K E 13 165 P I F G E IL E L 13 208 N R F Y S H H R A 13 5 G D S D G V N L I 12 78 G S G I I LV G K F 12 116 I K L L V R V Y V 12 120 V R V Y V V K A T 12 137 G K A DP Y V V V 12 151 R Q D T K E R Y I 12 156 E R Y I P K Q L N 12 169 E I LE L S I S L 12 190 H D L V G S D D L 12 11 N L I S M V G E I 11 23 G E AE V K G T V 11 32 S P K K A V A T L 11 34 K K A V A T L K I 11 55 E D WL N V F P L 11 91 L I Y P E S E A V 11 92 I Y P E S E A V L 11 93 Y P ES E A V L F 11 107 S R G I P Q N R P 11 110 I P Q N R P I K L 11 112 Q NR P I K L L V 11 113 N R P I K L L V R 11 150 E R Q D T K E R Y 11 179 AE T E L T V A V 11 214 H R A N C G L A S 11 218 C G L A S Q Y E V 11 39T L K I Y N R S L 10 44 N R S L E E E F N 10 50 E F N H F E D W L 10 64Y R G Q G G Q D G 10 83 V G K F K G S F L 10 111 P Q N R P I K L L 10136 N G K A D P Y V V 10 159 I P K Q L N P I F 10 162 Q L N P I F G E I10 163 L N P I F G E I L 10 184 T V A V F E H D L 10 196 D D L I G E T HI 10 198 L I G E T H I D L 10 202 T H I D L E N R F 10 212 S H H R A N CG L 10 2 D D P G D S D G V 9 8 D G V N L I S M V 9 19 I Q D Q G E A E V9 43 Y N R S L E E E F 9 102 S E P Q I S R G I 9 135 P N G K A D P Y V 9167 F G E I L E L S I 9 177 L P A E T E L T V 9 180 E T E L T V A V F 9185 V A V F E H D L V 9 191 D L V G S D D L I 9 220 L A S Q Y E V W V 97 S D G V N L I S M 8 46 S L E E E F N H F 8 53 H F E D W L N V F 8 75 EE E G S G H L V 8 82 L V G K F K G S F 8 157 R Y I P K Q L N P 8 158 Y IP K Q L N P I 8 228 V Q Q G P Q E P F 8 45 R S L E E E F N H 7 88 G S FL I Y P E S 7 209 R F Y S H H R A N 7 HLA-B*5101 nonamers 177 L P A E TE L T V 26 110 I P Q N R P I K L 22 114 R P I K L L V R V 22 140 D P Y VV V S A G 22 220 L A S Q Y E V W V 22 32 S P K K A V A T L 21 136 N G KA D P Y V V 20 3 D P G D S D G V N 19 8 D G V N L I S M V 19 185 V A V FE H D L V 19 108 R G I P Q N R P I 18 167 F G E I L E L S I 18 196 D D LI G E T H I 18 218 C G L A S Q Y E V 18 134 D P N G K A D P Y 17 138 K AD P Y V V V S 17 130 L A P A D P N G K 16 158 Y I P K Q L N P I 16 178 PA E T E L T V A 16 191 D L V G S D D L I 16 24 E A E V K G T V S 15 92 IY P E S E A V L 15 116 I K L L V R V Y V 15 117 K L L V R V Y V V 15 2 DD P G D S D G V 14 5 G D S D G V N L I 14 11 N L I S M V G E I 14 23 G EA E V K G T V 14 35 K A V A T L K I Y 14 83 V G K P K G S F L 14 91 L IY P E S E A V 14 93 Y P E S E A V L F 14 131 A P A D P N G K A 14 4 P GD S D G V N L 13 34 K K A V A T L K I 13 37 V A T L K I Y N R 13 52 N HF E D W L N V 13 61 F P L Y R G Q G G 13 98 A V L F S E P Q I 13 137 G KA D P Y V V V 13 151 R Q D T K E R Y I 13 159 I P K Q L N P I F 13 part3: MHC Class I nonamer analysis of 158P3D2 v.3 (aa 95-111,PTEREVSVRRRSGPFAL). Pos 1 2 3 4 5 6 7 8 9 score HLA-A1 nonamers 95 P T ER E V S V R 18 HLA-A26 nonamers 101 S V R R R S G P F 20 99 E V S V R RR S G 17 94 P T E R E V S V R 15 HLA-A3 nonamers 101 S V R R R S G P F24 99 E V S V R R R S G 15 HLA-B*0702 nonamers 103 R R R S G P F A L 14102 V R R R S G P F A 11 HLA-B*08 nonamers 101 S V R R R S G P F 22 103R R R S G P F A L 17 HLA-B*1510 nonamers 103 R R R S G P F A L 13 96 E RE V S V R R R 8 HLA-B*2705 nonamers 103 R R R S G P F A L 26 96 E R E VS V R R R 24 95 T E R E V S V R R 16 94 P T E R E V S V R 14 HLA-B*2709nonamers 103 R R R S G P F A L 25 part 4: MHC Class I nonamer analysisof 158P3D2 v.4 (aa 94-110, LPTEREVSIWRRSGPFA). Pos 1 2 3 4 5 6 7 8 9score HLA-A*0201 nonamers 94 L P T E R E V S I 15 HLA-A1 nonamers 95 PT E R E V S I W 17 HLA-A26 nonamers 101 S I W R R S G P F 20 99 E V S IW R R S G 17 95 P T E R E V S I W 15 HLA-A3 nonamers 101 S I W R R S G PF 19 HLA-B*0702 nonamers 94 L P T E R E V S I 18 102 I W R R S G P F A12 HLA-B*08 nonamers 94 L P T E R E V S I 23 101 S I W R R S G P F 20HLA-B*2705 nonamers 4 E R E V S I W R R 27 3 T E R E V S I W R 14HLA-B*5101 nonamers 94 L P T E R E V S I 25 part 5: MHC Class I nonameranalysis of 158P3D2 v.5a (aa 122-178, LVLQVWDYTASLPMTSLDPWSCSYQTWCVGPGAPSSALC SWPAMGPGRG AICFAAAA) Pos 1 2 3 4 5 6 7 8 9 score HLA-A*0201nonamers 125 Q V W D Y T A S L 23 164 A M G P G R G A I 23 123 V L Q V WD Y T A 18 130 T A S L P M T S L 17 137 S L D P W S C S Y 16 170 G A I CF A A A A 15 157 S A L C S W P A M 14 158 A L C S W P A M G 14 132 S L PM T S L D P 13 142 S C S Y Q T W C V 13 151 G P G A P S S A L 13 121 L VL Q V W D Y T 12 129 Y T A S L P M T S 11 146 Q T W C V G P G A 11 149 GV G P G A P S S 11 HLA-A1 nonamers 16 S L D P W S C S Y 32 HLA-A26noamers 16 S L D P W S C S Y 22 4 Q V W D Y T A S L 21 8 Y T A S L P M TS 14 28 C V G P G A P S S 14 7 D Y T A S L P M T 13 9 T A S L P M T S L13 25 Q T W C V G P G A 12 1 L V L Q V W D Y T 11 14 M T S L D P W S C11 36 S A L C S W P A M 11 37 A L C S W P A M G 11 HLA-A3 nonamers 16 SL D P W S C S Y 22 28 G V G P G A P S S 20 4 Q V W D Y T A S L 18 37 A LC S W P A M G 18 2 V L Q V W D Y T A 14 11 S L P M T S L D P 14 1 L VL Q V W D Y T 13 166 G P G R G A I C F 12 131 A S L P M T S L D 11 164 AM G P G R G A I 11 HLA-B*0702 nonamers 151 G P G A P S S A L 26 166 G PG R G A I C F 17 130 T A S L P M T S L 16 139 D P W S C S Y Q T 16 154 AP S S A L C S W 14 163 P A M G P G R G A 13 HLA-B*08 nonamers 151 G P GA P S S A L 18 130 T A S L P M T S L 15 166 G P G R G A I C F 13 125 Q VW D Y T A S L 10 HLA-B*1510 nonamers 130 T A S L P M T S L 14 151 G P GA P S S A L 13 125 Q V W D Y T A S L 11 157 S A L C S W P A M 8 166 G PG R G A I C F 8 HLA-B*2705 nonamers 166 G P G R G A I C F 17 151 G P G AP S S A L 16 130 T A S L P M T S L 15 168 G R G A I C F A A 14 125 Q V WD Y T A S L 12 127 W D Y T A S L P M 12 157 S A L C S W P A M 12 137 S LD P W S C S Y 11 161 S W P A M G P G R 11 164 A M G P G R G A I 10HLA-B*2709 nonamers 168 G R G A I C F A A 14 151 G P G A P S S A L 13146 G P G R G A I C F 12 127 W D Y T A S L P M 11 157 S A L C S W P A M11 125 Q V W D Y T A S L 10 130 T A S L P M T S L 10 161 A M G P G R G AI 10 142 S C S Y Q T W C V 8 HLA-B*4402 nonamers 164 A M G P G R G A I17 137 S L D P W S C S Y 15 154 A P S S A L C S W 15 133 L P M T S L D PW 13 166 G P G R G A I C F 13 125 Q V W D Y T A S L 12 130 T A S L P M TS L 12 140 P W S C S Y Q T W 12 151 G P G A P S S A L 12 131 A S L P M TS L D 9 HLA-B*5101 nonamers 130 T A S L P M T S L 18 151 G P G A P S S AL 17 133 L P M T S L D P W 15 139 D P W S C S Y Q T 15 153 G A P S S A LC S 14 157 S A L C S W P A M 13 162 W P A M G P G R G 12 163 P A M G P GR G A 12 166 G P G R G A I C F 12 154 A P S S A L C S W 11 170 G A I C FA A A A 11 150 V G P G A P S S A 10 164 A M G P G R G A I 10 125 Q V W DY T A S L 9 165 M G P G R G A I C 9

[0778] TABLE XIXB MHC Class I Analysis of 158P3D2 (decamers) part 1: MHCClass I decamer analysis of 158P3D2 v.1 (aa 1-328) Listed are scoreswhich correlate with the ligation strength to a defined HLA type for asequence of amino acids. The algorithms used are based on the book “MHCLigands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S.Stevanovic. The probability of being processed and presented is given inorder to predict T-cell epitopes. Pos 1 2 3 4 5 6 7 8 9 0 scoreHLA-A*0201 decamers 296 V L L L L V L L T V 30 301 V L L T V F L L L V28 93 Y L P T E R E V S V 26 294 T L V L L L L V L L 26 298 L L L V LL T V F L 26 299 L L V L L T V F L L 26 312 Y T I P G Q I S Q V 24 151 GA R G P E L C S V 23 31 V I W N T E D V V L 22 236 V E A E F E L L T V22 286 F I W R R Y W R T L 22 140 S L E L Q L P D M V 21 293 R T L V LL L L V L 21 132 I S A N D F L G S L 20 179 L R G W W P V V K L 20 123 VL Q V W D Y D R I 19 148 M V R G A R G P E L 19 300 L V L L T V F L L L19 223 T D M G G N V Y I L 18 297 L L L L V L L T V F 18 313 T I P G QI S Q V I 18 317 Q I S Q V I F R P L 18 30 V V I W N T E D V V 17 230 YI L T G K V E A E 17 231 I L T G K V E A E F 17 289 R R Y W R T L V L L17 291 Y W R T L V L L L L 17 295 L V L L L L V L L T 17 308 L L V F YT I P G Q 17 4 D I F P Q D V P A P 16 22 QP I S Y E L R V V 16 45 P L TG E M S S D I 16 54 I Y V K S W V K G L 16 187 K L K E A E D V E R 16271 T S F N W F V N P L 16 290 R Y W R T L V L L L 16 302 L L T V F L LL V F 16 39 V L D D E N P L T G 15 114 A E F R Q P A V L V 15 117 R Q PA V L V L Q V 15 137 F L G S L E L Q L P 15 143 L Q L P D M V R G A 15222 F T D M G G N V Y I 15 244 T V E E A E K R P V 15 HLA-A*0203decamers 194 V E R E A Q E A Q A 18 3 I D I F P Q D V P A 10 101 S V W RR S G P F A 10 105 R S G P F A L E E A 10 111 L E E A E F R Q P A 10 125Q V W D Y D R I S A 10 143 L Q L P D M V R G A 10 154 G P E L C S V Q LA 10 158 C S V Q L A R N G A 10 182 W W P V V K L K E A 10 189 K E A E DV E R E A 10 192 E D V E R E A Q E A 10 229 V Y I L T G K V E A 10 239 EF E L L T V E E A 10 4 D I F P Q D Y P A P 9 102 V W R R S G P F A L 9106 S G P F A L E E A E 9 112 E E A E F R Q P A V 9 126 V W D Y D R I SA N 9 144 Q L P D M V R G A R 9 155 P E L C S V Q L A R 9 159 S V Q L AR N G A G 9 183 W P V V K L K E A E 9 190 E A E D V E R E A Q 9 193 DV E R E A Q E A Q 9 195 E R E A Q E A Q A G 9 230 Y I L T G K V E A E 9240 F E L L T V E E A E 9 HLA-A1 decamers 17 D I K P R Q P I S Y 23 46 LT G E M S S D I Y 21 303 L T V F L L L V F Y 21 69 E T D V H F N S L T19 39 V L D D E N P L T G 18 222 F T D M G G N V Y I 18 235 K V E A E FE L L T 18 25 S Y E L R V V I W N 17 120 A V L V L Q V W D Y 17 134 AN D F L G S L E L 17 221 E F T D M G G N V Y 17 34 N T E D V V L D D E16 51 S S D I Y V K S W V 16 84 N W R F V F R F D Y 16 95 P T E R E VS V W R 16 110 A L E E A E F R Q P 15 282 T F V F F I W R R Y 15 47 TG E M S S D I Y V 14 140 S L E L Q L P D M V 14 198 A Q E A Q A G K K K14 259 Q P E P L E K P S R 14 262 P L E K P S R P K T 14 154 G P E L C SV Q L A 13 216 R P E D L E F T D M 13 245 V E E A E K R P V G 13 247 EA E K R P V G K G 13 293 R T L V L L L L V L 13 78 T G E G N F N W R F12 181 G W W P V V K L K E 12 312 Y T I P G Q I S Q V 12 2 W I D I F PQ D V P 11 15 P V D I K P R Q P I 11 62 G L E H D K Q E T D 11 64 E H DK Q E I D V H 11 111 L E E A E F R Q P A 11 113 E A E F R Q P A V L 11126 V W D Y D R I S A N 11 145 L P D M V R G A R G 11 166 G A G P R CN L F R 11 188 L K E A E D V E R E 11 190 E A E D V E R E A Q 11 191 AE D V E R E A Q E 11 217 P E D L E F T D M G 11 219 D L E F T D M G G N11 237 E A E F E L L T V E 11 239 E F E L L T V E E A 11 300 L Y L L T VF L L L 11 HLA-A26 decamers 17 D I K P R Q P I S Y 30 4 D I F P Q D V PA P 26 115 E F R Q P A V L V L 25 303 L T V F L L L V F Y 25 37 D V V LD D E N P L 24 120 A V L V L Q V W D Y 24 136 D F L G S L E L Q L 23 221E F T D M G G N V Y 23 317 Q I S Q V I F R P L 23 46 L T G E M S S D I Y22 148 M V R G A R G P E L 22 231 I L T G K V E A E F 22 276 F V N P LKT F V F 22 293 R T L V L L L L V L 22 297 L L L L V L L T V F 22 300 LV L L T V F L L L 22 302 L L T V F L L L V F 22 71 D V H F N S L T G E21 82 N F N W R F V F R F 21 156 E L C S V Q L A R N 21 294 T L V L L LL V L L 21 142 E L Q L P D M V R G 20 299 L L V L L T V F L L 20 312 Y TI P G Q I S Q V 20 31 V I W N T E D V V L 19 219 D L E F T D M G G N 19264 E K P S R P K T S F 19 9 D V P A P P P V D I 18 53 D I Y V K S W V KG 18 69 E T D V H F N S L T 18 99 E V S V W R R S G P 18 286 F I W R R YW R T L 18 128 D Y D R I S A N D F 17 193 D V E R E A Q E A Q 17 239 E FE L L T V E E A 17 281 K T F V F F I W R R 17 282 T F V F F I W R R Y 17298 L L L V L L T V F L 17 77 L T G E G N F N W R 16 80 E G N F N W R FV F 16 87 F V F R P D Y L P T 16 241 E L L T V E E A E K 16 267 S R P KT S F N W F 16 270 K T S F N W F V N P 16 274 N W F V N P L K T F 16 277V N P L K T F V F F 16 304 T V F L L L V F Y T 16 34 N T E D V V L D D E15 41 D D E N P L T G E M 15 110 A L E E A E F R Q P 15 113 E A E F R QP A V L 15 131 R I S A N D F L G S 15 139 G S L E L Q L P D M 15 230 Y IL T G K V E A E 15 HLA-A3 decamers 178 R L R G W W P V V K 38 241 E LL T V E E A E K 25 161 Q L A R N G A G P R 24 187 K L K E A E D V E R 232281 N V Y I L T G K V E 22 297 L L L L V L L T V F 22 17 D I K P RQ P I S Y 21 120 A V L V L Q V W D Y 21 231 I L T G K V E A E F 21 276 FV N P L K T F V F 21 302 L L T V F L L L V F 21 144 Q L P D M V R G A R20 296 V L L L L V L L T V 20 39 V L D D E N P L T G 19 HLA-B*0702decamers 19 K P R Q P I S Y E L 23 314 I P G Q I S Q V I F 20 216 R P ED L E F T D M 19 278 N P L K T F V P F I 19 107 G P F A L E E A E F 18268 R P K T S F N W F V 18 22 Q P I S Y E L R V V 17 115 E F R Q P A V LV L 17 154 G P E L C S V Q L A 17 288 W R R Y W R T L V L 16 134 A N D FL G S L E L 15 148 M V R G A R G P E L 15 265 K P S R P K T S F N 15 12A P P P V D I K P R 14 136 D F L G S L E L Q L 14 164 R N G A G P R C NL 14 179 L R G W W P V V K L 14 211 R R R K G R P E D L 14 223 T D M G GN V Y I L 14 251 R P V G K G R K Q P 14 290 R Y W R T L V L L L 14 291 YW R T L V L L L L 14 293 R T L V L L L L V L 14 298 L L L V L L T V F L14 317 Q I S Q V I F R P L 14 10 V P A P P P V D I K 13 31 V I W N T E DV V L 13 54 I Y V K S W V K G L 13 102 V W R R S G P F A L 13 113 E A EF R Q P A V L 13 129 Y D R I S A N D F L 13 153 R G P E L C S V Q L 13168 G P R C N L F R C R 13 289 R R Y W R T L V L L 13 300 L V L L T V FL L L 13 6 F P Q D V P A P P P 12 23 P I S Y E L R V V I 12 94 L P T E RE V S V W 12 118 Q P A V L V L Q V W 12 132 I S A N D F L G S L 12 145 LP D M V R G A R G 12 254 G K G R K Q P E P L 12 259 Q P E P L E K P S R12 261 E P L E K P S R P K 12 271 T S F N W F V N P L 12 294 T L V L L LL V L L 12 HLA-B*4402 decamers 68 Q E T D V H F N S L 23 114 A E F R Q PA V L V 19 238 A E F E L L T V E E 17 263 L E K P S R P K T S 17 274 N WF V N P L K T F 17 248 A E K R P V G K G R 16 17 D I K P R Q P I S Y 1526 Y E L R V V I W N T 15 48 G E M S S D I Y V K 15 50 M S S D I Y V K SW 15 115 E F R Q P A V L V L 15 120 A V L V L Q V W D Y 15 134 A N D F LG S L E L 15 191 A E D V E R E A Q E 15 221 E F T D M G G N V Y 15 271 TS F N W F V N P L 15 276 F V N P L K T F V F 15 300 L V L L T V F L L L15 80 E G N F N W R F V F 14 102 V W R R S G P F A L 14 112 E E A E F RQ P A V 14 113 E A E F R Q P A V L 14 128 D Y D R I S A N D F 14 136 D FL G S L E L Q L 14 155 P E L C S V Q L A R 14 165 N G A G P R C N L F 14240 F E L L T V E E A E 14 246 E E A E K R P V G K 14 267 S R P K T S FN W F 14 277 V N P L K T F V F F 14 283 F V F F I W R R Y W 14 290 R Y WR T L V L L L 14 291 Y W R T L V L L L L 14 293 R T L V L L L L V L 14294 T L V L L L L V L L 14 297 L L L L V L L T V F 14 31 V I W N T E D VV L 13 42 D E N P L T G E M S 13 54 I Y V K S W V K G L 13 85 W R F V FR F D Y L 13 153 R G P E L C S V Q L 13 179 L R G W W P V V K L 13 199 QE A Q A G K K K R 13 217 P E D L E F T D M G 13 220 L E F T D M G G N V13 223 T D M G G N V Y I L 13 236 V E A E F E L L T V 13 260 P E P L E KP S R P 13 264 E K P S R P K T S F 13 266 P S R P K T S F N W 13 286 F IW R R Y W R T L 13 288 W R R Y W R T L V L 13 289 R R Y W R T L V L L 13298 L L L V L L T V F L 13 299 L L V L L T V F L L 13 302 L L T V F L LL V F 13 317 Q I S Q V I F R P L 13 12 A P P P V D I K P R 12 23 P I S YE L R V V I 12 24 I S Y E L R V V I W 12 37 D V V L D D E N P L 12 74 FN S L T G E G N F 12 76 S L T G E G N F N W 12 79 G E G N F N W R F V 1282 N F N W R F V F R F 12 84 N W R F V F R F D Y 12 94 L P T E R E V S VW 12 98 R E V S V W R R S G 12 107 G P F A L E E A E F 12 118 Q P A V LV L Q V W 12 132 I S A N D F L G S L 12 141 L E L Q L P D M V R 12 170 RC N L F R C R R L 12 173 L F R C R R L R G W 12 174 F R C R R L R G W W12 189 K E A E D V E R E A 12 213 R K G R P E D L E F 12 234 G K V E A EF E L L 12 245 V E E A E K R P V G 12 254 G K G R K Q P E P L 12 279 P LK T F V F F I W 12 303 L T V F L L L V F Y 12 305 V F L L L V F Y T I 12309 L V F Y T I P G Q I 12 9 D V P A P P P V D I 11 19 K P R Q P I S Y EL 11 35 T E D V V L D D E N 11 63 L E H D K Q E T D V 11 65 H D K Q E TD V H F 11 78 T G E G N F N W R F 11 96 T E R E V S V W R R 11 111 L E EA E F R Q P A 11 148 M V R G A R G P E L 11 164 R N G A G P R C N L 11194 V E R E A Q E A Q A 11 211 R R R K G R P E D L 11 231 I L T G K V EA E F 11 278 N P L K T F V F F I 11 282 T F V F F I W R R Y 11 313 T I PG Q I S Q V I 11 314 I P G Q I S Q V I F 11 part 2: MHC Class I decameranalysis of 158P3D2 v.2a (aa 1-236). HLA-A*0201 decamers 91 L I Y P ES E A V L 25 176 S L P A E T E L T V 25 219 G L A S Q Y E V W V 25 118 LL V R V Y V V K A 24 90 F L I Y P E S E A V 23 162 Q L N P I F G E I L23 197 D L I G E T H I D L 23 174 S I S L P A E T E L 22 109 G I P Q NE P I K L 21 18 E I Q D Q E A E V 20 38 A T L K I Y N R S L 20 31 V S PK K A V A T L 19 116 I K L L V R V Y V V 19 138 K A D P Y V V V S A 19164 N P I F G E I L E L 18 10 V N L I S M V G E I 17 157 R Y I P K Q L NP I 17 183 L T V A V F E H D L 17 7 S D G V N L I S M V 16 41 KI Y N RS L E E E 16 57 W L N V F P L Y R G 16 82 L V G K F K G S F L 16 113 N RP I K L L V R V 16 115 P I K L L V R V Y V 16 129 N L A P A D P N G K 16170 I L E L S I S L P A 16 184 T V A V F E H D L V 16 186 A V F E H D LV G S 16 198 L I G E T H I D L E 16 54 F E D W L N V F P L 15 110 I P QN R P I K L L 15 117 K L L V R V Y V V K 15 121 R V Y V V K A T N L 15166 I F G E I L E L S I 15 168 G E I L E L S I S L 15 172 E L S I S L PA E T 15 46 S L E E E F N H F E 14 81 H L V G K F K G S F 14 99 V L F SE P Q I S R 14 119 L V R V Y V V K A T 14 124 V V K A T N L A P A 14 177L P A E T E L T V A 14 1 M D D P G D S D G V 13 30 T V S P K K A V A T13 36 A V A T L K I Y N R 13 74 G E E E G S G H L V 13 134 D P N G K A DP Y V 13 161 K Q L N P I F G E I 13 169 E I L E L S I S L P 13 178 P A ET E L T V A V 13 211 Y S H H R A N C G L 13 217 N C G L A S Q Y E V 13 3D P G D S D G V N L 12 11 N L I S M V G E I Q 12 14 S M V G E I Q D Q G12 73 G G E E E G S G H L 12 130 L A P A D P N G K A 12 165 P I F G EI L E L S 12 191 D L V G S D D L I G 12 193 V G S D D L I G E T 12HLA-A*0203 decamers 29 G T V S P K K A V A 18 124 V V K A T N L A P A 1816 V G E I Q D Q G E A 10 27 V K G T V S P K K A 10 89 S F L I Y P E S EA 10 118 L L V R V Y V V K A 10 122 V Y V V K A T N L A 10 130 L A P A DP N G K A 10 138 K A D P Y V V V S A 10 170 I L E L S I S L P A 10 177 LP A E T E L T V A 10 207 E N R F Y S H H R A 10 212 S H H R A N C G L A10 17 G E I Q D Q G E A E 9 28 K G T V S P K K A V 9 30 T V S P K K A VA T 9 90 F L I Y P E S E A V 9 119 L V R V Y V V K A T 9 123 Y V V K A TN L A P 9 125 V K A T N L A P A D 9 131 A P A D P N G K A D 9 139 A D PY V V V S A G 9 171 L E L S I S L P A E 9 178 P A E T E L T V A V 9 208N R F Y S H H R A N 9 213 H H R A N C G L A S 9 HLA-A1 decamers 84 G K FK G S F L I Y 24 55 E D W L N V F P L Y 20 6 D S D G V N L I S M 19 93 YP E S E A V L F S 19 101 F S E P Q I S R G I 19 75 E E E G S G H L V G18 95 E S E A V L F S E P 17 114 R P I K L L V R V Y 17 170 I L E L S IS L P A 17 133 A D P N G K A D P Y 16 180 E T E L T V A V F E 16 199 IG E T H I D L E N 16 202 T H I D L E N R F Y 16 34 K K A V A T L K I Y15 138 K A D P Y V V V S A 15 149 R E R Q D T K E R Y 15 194 G S D D L IG E T H 15 215 R A N C G L A S Q Y 15 1 M D D P G D S D G V 14 46 S L EE E F N H F E 14 132 P A D P N G K A D P 14 4 P G D S D G V N L I 13 48E E E F N H F E D W 13 74 G E E E G S G H L V 13 54 F E D W L N V F P L12 187 V F E H D L V G S D 12 195 S D D L I G E T H I 12 HLA-A26decamers 201 E T H I D L E N R F 27 197 D L I G E T H I D L 26 153 D T KE R Y I P K Q 25 77 E G S G H L V G K F 23 158 Y I P K Q L N P I F 23169 E I L E L S I S L P 23 6 D S D G V N L I S M 22 26 E V K G T V S P KK 21 55 E D W L N V F P L Y 21 81 HL V G K F K G S F 21 91 L I Y P E S EA V L 21 109 G I P Q N R P I K L 21 227 W V Q Q G P Q E P F 21 38 A T LK I Y N R S L 20 82 L V G K F K G S F L 20 186 A V F E H D L V G S 20205 D L E N R F Y S H H 20 18 E I Q D Q G E A E V 19 121 R V Y V V K A TN L 19 174 S I S L P A E T E L 19 182 E L T V A V F E H D 19 52 N H F ED W L N V F 18 162 Q L N P I F G E I L 18 165 P I F G E I L E L S 18 183L T V A V F E H D L 18 225 E V W V Q Q G P Q E 18 3 D P G D S D G V N L17 45 R S L E E E F N H P 17 84 G K F K G S F L I Y 17 114 R P I K L L VR V Y 17 179 A E T E L T V A V F 17 180 E T E L T V A V F E 17 9 G V N LI S M V G E 16 36 A V A T L K I Y N R 16 49 E E F N H F E D W L 16 124 VV K A T N L A P A 16 172 E L S I S L P A E T 16 191 D L V G S D D L I G16 192 L V G S D D L I G E 16 198 L I G E T H I D L E 16 31 V S P K K AV A T L 15 34 K K A V A T L K I Y 15 41 K I Y N R S L E E E 15 59 N V FP L Y R G Q G 15 119 L V R V Y V V K A T 15 164 N P I F G E I L E L 15189 E H D L V G S D D L 15 15 M V G E I Q D Q G E 14 30 T V S P K K A VA T 14 92 I Y P E S E A V L F 14 100 L F S E P Q I S R G 14 118 L L V RV Y V V K A 14 21 D Q G E A E V K G T 13 54 F E D W L N V F P L 13 57 WL N V F P L Y R G 13 76 E E G S G H L V G K 13 85 K F K G S F L I Y P 13117 K L L V R V Y V V K 13 202 T H I D L E N R F Y 13 HLA-A3 decamers117 K L L V R V Y V V K 33 129 N L A P A D P N G K 25 62 P L Y R G Q G QD 24 26 E V K G T V S P K K 23 91 L I Y P E S E A V L 22 121 R V Y V V KA T N L 22 30 T V S P K K A V A T 21 108 R G I P Q N R P I K 21 176 S LP A E T E L T V 20 19 I Q D Q G E A E V K 19 81 H L V G K F K G S F 19112 Q N R P I K L L V R 19 215 R A N C G L A S Q Y 19 36 A V A T L K I YN R 18 59 N V F P L Y R G Q G 18 105 Q I S R G I P Q N R 18 146 S A G RE R Q D T K 18 162 Q L N P I F G E I L 18 186 A V F E H D L V G S 18 205D L E N R F Y S H H 18 114 R P I K L L V R V Y 17 118 L L V R V Y V K A17 142 Y V V V S A G R E R 17 11 N L I S M V G E I Q 16 25 A E V K GT V S P K 16 32 S P K K A V A T L K 16 39 T L K I Y N R S L E 16 41 K IY N R S L E E E 16 82 L V G K F K G S F L 16 98 A V L F S E P Q I S 1699 V L F S E P Q I S R 16 124 V V K A T N L A P A 16 170 I L E L S I S LP A 16 219 G L A S Q Y E V W V 16 225 E V W V Q Q Q P Q E 16 HLA-B*0702decamers 3 D P G D S D G V N L 23 164 N P I F G E I L E L 22 110 I P Q NR P I K L L 21 134 D P N G K A D P Y V 19 177 L P A E T E L T V A 19 93Y P E S E A V L F S 14 1114 R P I K L L V R V Y 14 131 A P A D P N G K AD 14 31 V S P K K A V A T L 13 38 A T L K I Y N R S L 13 54 F E D W L NV F P L 13 82 L V G K F K G S F L 13 91 L I Y P E S E A V L 13 174 S I SL P A E T E L 13 30 T V S P K K A V A T 12 32 S P K K A V A T L K 12 77E G S G H L V G K F 12 103 E P Q I S R G I P Q 12 121 R V Y V V K A T NL 12 138 K A D P Y V V V S A 12 159 I P K Q L N P I F G 12 189 E H D L VG S D D L 12 197 D L I G E T H I D L 12 49 E E F N H F E D W L 11 140 DP Y V V V S A G R 11 162 Q L N P I F G E I L 11 179 A E T E L T V A V F11 183 L T V A V F E H D L 11 HLA-B*4402 decamers 49 E E F N H F E D W L25 168 G E I L E L S I S L 25 179 A E T E L T V A V F 25 48 E E E F N HF E D W 23 54 F E D W L N V F P L 22 149 R E R Q D T K E R Y 20 164 N PI F G E I L E L 18 110 I P Q N R P I K L L 17 52 N H F E D W L N V F 1677 E G S G H L V G K F 16 114 R P I K L L V R V Y 16 133 A D P N G K A DP Y 16 157 R Y I P K Q L N P I 16 17 G E I Q D Q G E A E 15 38 A T L K IY N R S L 15 55 E D W L N V F P L Y 15 75 E E E G S G H L V G 15 154 T KE R Y I P K Q L 15 197 D L I G E T H I D L 15 202 T H I D L E N R F Y 1525 A E V K G T V S P K 14 34 K K A V A T L K I Y 14 76 E E G S G H L V GK 14 84 G K F K G S F L I Y 14 91 L I Y P E S E A V L 14 92 I Y P E S EA V L F 14 109 G I P Q N R P I K L 14 171 L E L S I S L P A E 14 189 E HD L V G S D D L 14 31 V S P K K A V A T L 13 45 R S L E E E F N H F 13102 S E P Q I S R G I P 13 162 Q L N P I F G E I L 13 174 S I S L P A ET E L 13 181 T E L T V A V F E H 13 201 E T H I D L E N R F 13 3 D P G DS D G V N L 12 4 P G D S D G V N L I 12 74 G E E E G S G H L V 12 94 P ES E A V L F S E 12 97 E A V L F S E P Q I 12 101 F S E P Q I S R G I 12150 E R Q D T K E R Y I 12 155 K E R Y I P K Q L N 12 161 K Q L N P I FG E I 12 206 L E N R F Y S H H R 12 215 R A N C G L A S Q Y 12 218 C G LA S Q Y E V W 12 part 3: MHC Class I decamer analysis of 158P3D2 v.3 ,(aa 94-103-112, LPTEREVSVRRRSGPFALE). HLA-A*0203 decamers 101 S V R R RS G P F A 10 102 V R R R S G P F A L 9 HLA-A1 decamers 95 P T E R E VS V R R 16 97 E R E V S V R R R S 11 HLA-A26 decamers 99 E V S V R R R SG P 18 HLA-A3 decamers 101 S V R R R S G P F A 20 99 E V S V R R R S G P14 HLA-B*0702 decamers 102 V R R R S G P F A L 13 94 L P T E R E V S V R12 HLA-B*4402 decamers 102 V R R R S G P F A L 14 96 T E R E V S V R R R12 98 R E V S V R R R S G 12 100 V S V R R R S G P F 11 part 4: MHCClass I decamer analysis of 158P3D2 v.4 (aa 93-102-111,YLPTEREVSIWRRSGPFAL). Pos 1 2 3 4 5 6 7 8 9 0 score HLA-A*0201 decamers101 S I W R R S G P F A 16 HLA-A*0203 decamers 101 S I W R R S G P F A10 1021 I W R R S G P F A L 9 HLA-A1 decamers 95 P T E R E V S I W R 2097 E R E V S I W R R S 10 HLA-A26 decamers 99 E V S I W R R S G P 18HLA-B*0702 decamers 102 I W R R S G P F A L 14 HLA-B*4402 decamers 102 IW R R S G P F A L 14 95 T E R E V S I W R R 13 100 V S I W R R S G P F13 98 R E V S I W R R S G 12 94 L P T E R E V S I W 11 part 5: MHC ClassI decamer analysis of 158P3D2 via (aa 121-178,VLVLQVWDYTSLPMTSLDPWSCSYQTWCVGPGA PSSALCSWPAMGPGRGAICFAAAA). Pos 1 2 3 45 6 7 8 9 0 score HLA-A*0201 decamers 9 Y T A S L P M T S L 20 4 L Q V WD Y T A S L 16 12 S L P M T S L D P W 16 1 V L V L Q W D Y T 15 2 L V LQ V W D Y T A 15 17 S L D P W S C S Y Q 14 30 V G P G A P S S A L 14 44A M G P G R G A I C 14 38 A L C S W P A M G P 13 43 P A M G P G R G A I13 3 V L Q V W D Y T A S 12 29 C V G P G A P S S A 12 33 G A P S S A L CS W 11 7 W D Y T A S L P M T 10 21 W S C S Y Q T W C V 10 36 S S A L CS W P A M 10 37 S A L C S W P A M G 10 HLA-A*0203 decamers 48 G R G A IC F A A A 27 49 R G A I C F A A A A 27 47 P G R G A I C F A A 19 HLA-A1decamers 16 T S L D P W S C S Y 19 6 V W D Y T A S L P M 17 17 S L D P WS C S Y Q 17 11 A S L P M T S L D P 15 32 P G A P S S A L C S 10 40 CS W P A M G P G R 9 HLA-A26 decamers 9 Y T A S L P MT S L 25 29 C V G PG A P S S A 14 3 V L Q V W D Y T A S 13 12 S L P M T S L D P W 13 30 V GP G A P S S A L 13 45 M G P G R G A I C F 13 5 Q V W D Y T A S L P 12 16T S L D P W S C S Y 12 17 S L D P W S C S Y Q 12 19 D P W S C S Y Q T W12 HLA-A3 decamers 5 Q V W D Y T A S L P 19 29 C V G P G A P S S A 19 17S L D P W S C S Y Q 17 38 A L C S W P A M G P 17 2 L V L Q V W D Y T A16 49 R G A I C F A A A A 13 3 V L Q V W D Y T A S 12 44 A M G P G R G AI C 12 1 V L V L Q V W D Y T 11 11 A S L P M T S L D P 11 12 S L P M TS L D P W 11 16 T S L D P W S C S Y 11 32 P G A P S S A L C S 10 40 C SW P A M G P G R 10 45 M G P G R G A I C F 9 HLA-B*0702 decamers 46 G P GR G A I C F A 18 42 W P A M G P G R G A 17 34 A P S S A L C S W P 14 30V G P G A P S S A L 13 31 G P G A P S S A L C 13 4 L Q V W D Y T A S L12 9 Y T A S L P M T S L 12 13 L P M T S L D P W S 12 43 P A M G P G R GA I 11 47 P G R G A I C F A A 11 48 G R G A I C F A A A 11 6 V W D Y T AS L P M 10 19 D P W S C S Y Q T W 10 35 P S S A L C S W P A 10 49 R G AI C F A A A A 10 36 S S A L C S W P A M 9 HLA-B*4402 decamers 30 V G P GA P S S A L 14 45 M G P G R G A I C F 14 12 S L P M T S L D P W 13 43 PA M G P G R G A I 13 16 T S L D P W S C S Y 12 33 G A P S S A L C S W 124 L Q V W D Y T A S L 11 19 D P W S C S Y Q T W 11 9 Y T A S L P M T S L10 11 A S L P M T S L D P 8

[0779] TABLE XIXC MHC Class II Analysis of 158P3D2 part 1: MHC Class 1115-mer analysis of 1 58P3D2 v.1 (aa 1-328). Listed are scores whichcorrelate with the ligation strength to a defined HLA type for asequence of amino acids. The algorithms used are based on the book “MHCLigands and Peptide Motifs” by H.G.Ranimensee, J. Bachmann and S.Stevanovic. The probability of being processed and presented is given inorder to predict T-cell epitopes. Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5score HLA-DRB1*0101 15-mers 126 V W D Y D R I S A N D F L G S 32 308 L LV F Y T I P G Q I S Q V I 32 274 N W F V N P L K T F V F F I W 31 296 VL L L L V L L T V F L L L V 30 71 D V H F N S L T G E G N F N W 29 138 LG S L E L Q L P D M V R G A 29 226 G G N V Y I L T G K V E A E F 28 289R R Y W R T L V L L L L V L L 28 311 F Y T I P G Q I S Q V I F R P 28100 V S V W R R S G P F A L E E A 27 183 W P V V K L K E A E D V E R E27 237 E A E F E L L T V E E A E K R 27 303 L T V F L L L V F Y T I P GQ 27 27 E L R V V I W N T E D V V L D 26 146 P D M V R G A R G P E L C SV 26 173 L F R C R R L R G W W P V V K 26 219 D L E F T D M G G N V Y IL T 26 292 W R T L V L L L L Y L L T V F 26 297 L L L L V L L T V F L LL V F 26 40 L D D E N P L T G E M S S D I 25 135 N D F L G S L E L Q L PD M V 25 180 R G W W P V V K L K E A E D V 25 294 T L V L L L L V L L TV F L L 25 3 I D I F P Q D V P A P P P V D 24 52 S D I Y V K S W V K G LE H D 24 88 V F R F D Y L P T E R E V S V 24 99 E V S V W R R S G P F AL E E 24 132 I S A N D F L G S L E L Q L P 24 295 L V L L L L V L L T VF L L L 24 304 T V F L L L V F Y T I P G Q I 24 43 E N P L T G E M S S DI Y V K 23 2 W I D I F P Q D V P A P P P V 22 4 D I F P Q D V P A P P PV D I 22 7 P Q D V P A P P P V D I K P R 22 12 A P P P V D I K P R Q P IS Y 22 112 E E A E F R Q P A V L V L Q V 22 151 G A R G P E L C S V Q LA R N 22 225 M G G N V Y I L T G K V E A E 22 299 L L V L L T V F L L LV F Y T 22 307 L L L V F Y T I P G Q I S Q V 22 285 F F I W R R Y W R TL V L L L 21 84 N W R F V F R F D Y L P T E R 20 106 S G P F A L E E A EF R Q P A 20 113 E A E F R Q P A V L V L Q V W 20 144 Q L P D M V R G AR G P E L C 20 227 G N V Y I L T G K V E A E F E 20 273 F N W F V N P LK T F V F F I 20 13 P P P V D I K P R Q P I S Y E 19 57 K S W V K G L EH D K Q E T D 19 80 E G N F N W R F V F R F D Y L 19 82 N F N W R F V FR F D Y L P T 19 90 R F D Y L P T E R E V S V W R 19 156 E L C S V Q L AR N G A G P R 19 182 W W P V V K L K E A E D V E R 19 240 F E L L T V EE A E K R P V G 19 272 S F N W F V N P L K T F V F F 19 35 T E D V V L DD E N P L T G E 18 97 E R E V S V W R R S G P F A L 18 108 P F A L E E AE F R Q P A V L 18 129 Y D R I S A N D F L G S L E L 18 134 A N D F L GS L E L Q L P D M 18 158 C S V Q L A R N G A G P R C N 18 190 E A E D VE R E A Q E A Q A G 18 209 K Q R R R K G R P E D L E F T 18 214 K G R PE D L E F T D M G G N 18 242 L L T V E E A E K R P V G K G 18 288 W R RY W R T L V L L L L V L 18 29 R V V I W N T E D V V L D D E 17 34 N T ED V V L D D E N P L T G 17 47 T G E M S S D I Y V K S W V K 17 105 R S GP F A L E E A E F R Q P 17 159 S V Q L A R N G A G P R C N L 17 179 L RG W W P V V K L K E A E D 17 229 V Y I L T G K V E A E F E L L 17 230 YI L T G K V E A E F E L L T 17 284 V F F I W R R Y W R T L V L L 17 293R T L V L L L L V L L T V F L 17 298 L L L V L L T V F L L L V F Y 17300 L V L L T V F L L L V F Y T I 17 302 L L T V F L L L V F Y T I P G17 17 D I K P R Q P I S Y E L R V V 16 21 R Q P I S Y E L R V V I W N T16 25 S Y E L R V V I W N T E D V V 16 28 L R V V I W N T E D V V L D D16 91 F D Y L P T E R E V S V W R R 16 118 Q P A V L V L Q V W D Y D R I16 119 P A V L V L Q V W D Y D R I S 16 121 V L V L Q V W D Y D R I S AN 16 123 V L Q V W D Y D R I S A N D F 16 137 F L G S L E L Q L P D M VR G 16 176 C R R L R G W W P V V K L K E 16 196 R E A Q E A Q A G K K KR K Q 16 218 E D L E F T D M G G N V Y I L 16 261 E P L E K P S R P K TS F N W 16 281 K T F V F F I W R R Y W R T L 16 286 F I W R R Y W R T LV L L L L 16 290 R Y W R T L V L L L L V L L T 16 291 Y W R T L V L L LL V L L T V 16 HLA-DRB1*0301 (DR17) 15-mers 35 T E D V V L D D E N P L TG E 37 36 E D V V L D D E N P L T G E M 30 60 V K G L E H D K Q E T D VH F 27 134 A N D F L G S L E L Q L P D M 26 229 V Y I L T G K V E A E FE L L 26 47 T G E M S S D I Y V K S W V K 23 292 W R T L V L L L L V L LT V F 23 298 L L L V L L T V F L L L V F Y 23 295 L V L L L L V L L T VF L L L 22 296 V L L L L V L L T V F L L L V 22 297 L L L L V L L T V FL L L V F 21 300 L V L L T V F L L L V F Y T I 21 15 P V D I K P R Q P IS Y E L R 20 29 R V V I W N T E D V V L D D E 20 118 Q P A V L V L Q V WD Y D R I 20 239 E F E L L T V E E A E K R P V 20 274 N W F V N P L K TF V F F I W 20 3 I D I F P Q D V P A P P P V D 19 53 D I Y V K S W V K GL E H D K 19 74 F N S L T G E G N F N W R F V 19 86 R F V F R F D Y L PT E R E V 19 146 P D M V R G A R G P E L C S V 19 182 W W P V V K L K EA E D V E R 19 219 D L E F T D M G G N V Y I L T 19 13 P P P V D I K P RQ P I S Y E 18 21 R Q P I S Y E L R V V I W N T 18 113 E A E F R Q P A VL V L Q V W 18 130 D R I S A N D F L G S L E L Q 18 157 L C S V Q L A RN G A G P R C 18 213 R K G R P E D L E F T D M G G 18 233 T G K V E A EF E L L T V E E 18 242 L L T V E E A E K R P V G K G 18 260 P E P L E KP S R P K T S F N 18 284 V F F I W R R Y W R T L V L L 18 HLA-DRB1*0401(DR4Dw4) 15-mers 270 K T S F N W F V N P L K T F V 28 21 R Q P I S Y E LR V V I W N T 26 36 E D V V L D D E N P L T G E M 26 43 E N P L T G E MS S D I Y V K 26 57 K S W V K G L E H D K Q E T D 26 191 A E D V E R E AQ E A Q A G K 26 296 V L L L L V L L T V F L L L V 26 71 D V H F N S L TG E G N F N W 22 82 N F N W R F V F R F D Y L P T 22 88 V F R F D Y L PT E R E V S V 22 90 R F D Y L P T E R E V S V W R 22 124 L Q V W D Y D RI S A N D F L 22 180 R G W W P V V K L K E A E D V 22 237 E A E F E L LT V E E A E K R 22 285 F F I W R R Y W R T L V L L L 22 289 R R Y W R TL V L L L L V L L 22 303 L T V F L L L V F Y T I P G Q 22 308 L L V F YT I P G Q I S Q V I 22 309 L V F Y T I P G Q I S Q V I F 22 27 E L R V VI W N T E D V V L D 20 35 T E D V V L D D E N P L T G E 20 47 T G E M SS D I Y V K S W V K 20 60 V K G L E H D K Q E T D V H F 20 74 F N S L TG E G N F N W R F V 20 85 W R F V F R F D Y L P T E R E 20 91 F D Y L PT E R E V S V W R R 20 123 V L Q V W D Y D R I S A N D F 20 146 P D M VR G A R G P E L C S V 20 154 G P E L C S V Q L A R N G A G 20 157 L C SV Q L A R N G A G P R C 20 233 T G K V E A E F E L L T V E E 20 239 E FE L L T V E E A E K R P V 20 242 L L T V E E A E K R P V G K G 20 260 PE P L E K P S R P K T S F N 20 274 N W F V N P L K T F V F F I W 20 281K T F V F F I W R R Y W R T L 20 292 W R T L V L L L L V L L T V F 20293 R T L V L L L L V L L T V F L 20 294 T L V L L L L V L L T V F L L20 297 L L L L V L L T V F L L L V F 20 299 L L V L L T V F L L L V F YT 20 302 L L T V F L L L V F Y T I P G 20 305 V F L L L V F Y T I P G QI S 20 311 F Y T I P G Q I S Q V I F R P 20 50 M S S D I Y V K S W V K GL E 18 65 H D K Q E T D V H F N S L T G 18 109 F A L E E A E F R Q P A VL V 18 110 A L E E A E F R Q P A V L V L 18 132 I S A N D F L G S L E LQ L P 18 151 G A R G P E L C S V Q L A R N 18 156 E L C S V Q L A R N GA G P R 18 188 L K E A E D V E R E A Q E A Q 18 194 V E R E A Q E A Q AG K K K R 18 225 M G G N V Y I L T G K V E A E 18 3 I D I F P Q D V P AP P P V D 16 30 V V I W N T E D V V L D D E N 16 52 S D I Y V K S W V KG L E H D 16 56 V K S W V K G L E H D K Q E T 16 86 R F V F R F D Y L PT E R E V 16 100 V S V W R R S G P F A L E E A 16 106 S G P F A L E E AE F R Q P A 16 113 E A E F R Q P A V L V L Q V W 16 126 V W D Y D R I SA N D F L G S 16 134 A N D F L G S L E L Q L P D M 16 179 L R G W W P VV K L K E A E D 16 227 G N V Y I L T G K V E A E F E 16 273 F N W F V NP L K T F V F F I 16 280 L K T F V F F I W R R Y W R T 16 282 T F V F FI W R R Y W R T L V 16 288 W R R Y W R T L V L L L L V L 16 13 P P P V DI K P R Q P I S Y E 15 7 P Q D V P A P P P V D I K P R 14 25 S Y E L R VV I W N T E D V V 14 28 L R V V I W N T E D V V L D D 14 29 R V V I W NT E D V V L D D E 14 37 D V V L D D E N P L T G E M S 14 97 E R E V S VW R R S G P F A L 14 108 P F A L E E A E F R Q P A V L 14 118 Q P A V LV L Q V W D Y D R I 14 120 A V L V L Q V W D Y D R I S A 14 121 V L V LQ V W D Y D R I S A N 14 129 Y D R I S A N D F L G S L E L 14 135 N D FL G S L E L Q L P D M V 14 138 L G S L E L Q L P D M V R G A 14 142 E LQ L P D M V R G A R G P E 14 145 L P D M V R G A R G P E L C S 14 170 RC N L F R C R R L R G W W P 14 176 C R R L R G W W P V V K L K E 14 182W W P V V K L K E A E D V E R 14 185 V V K L K E A E D V E R E A Q 14217 P E D L E F T D M G G N V Y I 14 222 F T D M G G N V Y I L T G K V14 226 G G N V Y I L T G K V E A E F 14 240 F E L L T V E E A E K R P VG 14 277 V N P L K T F V F F I W R R Y 14 295 L V L L L L V L L T V F LL L 14 298 L L L V L L T V F L L L V F Y 14 300 L V L L T V F L L L V FY T I 14 304 T V F L L L V F Y T I P G Q I 14 306 F L L L V F Y T I P GQ I S Q 14 307 L L L V F Y T I P G Q I S Q V 14 HLA-DRB1*1101 15-mers179 L R G W W P V V K L K E A E D 27 90 R F D Y L P T E R E V S V W R 2582 N F N W R F V F R F D Y L P T 24 227 G N V Y I L T G K V E A E F E 24170 R C N L F R C R R L R G W W P 23 180 R G W W P V V K L K E A E D V23 308 L L V F Y T I P G Q I S Q V I 23 142 E L Q L P D M V R G A R G PE 22 237 E A E F E L L T V E E A E K R 22 281 K T F V F F I W R R Y W RT L 22 57 K S W V K G L E H D K Q E T D 21 96 T E R E V S V W R R S G PF A 21 97 E R E V S V W R R S G P F A L 21 123 V L Q V W D Y D R I S A ND F 20 156 E L G S V Q L A R N G A G P R 20 135 N D F L G S L E L Q L PD M V 19 219 D L E F T D M G G N V Y I L T 19 282 T F V F F I W R R Y WR T L V 19 285 F F I W R R Y W R T L V L L L 19 289 R R Y W R T L V L LL L V L L 19 304 T V F L L L V F Y T I P G Q I 19 3 I D I F P Q D V P AP P P V D 18 88 V F R F D Y L P T E R E V S V 18 273 F N W F V N P L K TF V F F I 18 303 L T V F L L L V F Y T I P G Q 18 53 D I Y V K S W V K GL E H D K 17 84 N W R F V F R F D Y L P T E R 17 65 H D K Q E T D V H FN S L T G 16 71 D V H F N S L T G E G N F N W 16 126 V W D Y D R I S A ND F L G S 16 167 A G P R C N L F R C R R L R G 16 204 G K K K R K Q R RR K G R P E 16 206 K K R K Q R R R K G R P E D L 16 247 E A E K R P V GK G R K Q P E 16 21 R Q P I S Y E L R V V I W N T 15 242 L L T V E E A EK R P V G K G 15 243 L T V E E A E K R P V G K G R 15 260 P E P L E K PS R P K T S F N 15 13 P P P V D I K P R Q P I S Y E 14 51 S S D I Y V KS W V K G L E H 14 109 F A L E E A E F R Q P A V L V 14 140 S L E L Q LP D M V R G A R G 14 143 L Q L P D M V R G A R G P E L 14 145 L P D M VR G A R G P E L C S 14 154 G P E L C S V Q L A R N G A G 14 188 L K E AE D V E R E A Q E A Q 14 249 E K R P V G K G R K Q P E P L 14 250 K R PV G K G R K Q P E P L E 14 257 R K Q P E P L E K P S R P K T 14 294 T LV L L L L V L L T V F L L 14 2 W I D I F P Q D V P A P P P V 13 12 A P PP V D I K P R Q P I S Y 13 25 S Y E L R V V I W N T E D V V 13 34 N T ED V V L D D E N P L T G 13 47 T G E M S S D I Y V K S W V K 13 108 P F AL E E A E F R Q P A V L 13 118 Q P A V L V L Q V W D Y D R I 13 226 G GN V Y I L T G K V E A E F 13 270 K T S F N W F V N P L K T F V 13 274 NW F V N P L K T F V F F I W 13 280 L K T F V F F I W R R Y W R T 13 292W R T L V L L L L V L L T V F 13 293 R T L V L L L L V L L T V F L 13295 L V L L L L V L L T V F L L L 13 296 V L L L L V L L T V F L L L V13 297 L L L L V L L T V F L L L V F 13 299 L L V L L T V F L L L V F YT 13 302 L L T V F L L L V F Y T I P G 13 305 V F L L L V F Y T I P G QI S 13 part 2: MHC Class 1115-met analysis of 158P3D2 v.2a (aa 1-236)Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score HLA-DRB1*0101 15-mers 119 L V RV Y V V K A T N L A P A 31 160 P K Q L N P I F G E I L E L S 31 61 F P LY R G Q G G Q D G G G E 27 90 F L I Y P E S E A V L F S E P 27 80 G H LV G K F K G S F L I Y P 26 113 N R P I K L L V R V Y V V K A 26 120 V RV Y V V K A T N L A P A D 25 139 A D P Y V V V S A G R E R Q D 25 96 S EA V L F S E P Q I S R G I 24 116 I K L L V R V Y V V K A T N L 24 156 ER Y I P K Q L N P I F G E I 24 164 N P I F G E I L E L S I S L P 24 167F G E I L E L S I S L P A E T 24 16 V G E I Q D Q G E A E V K G T 23 21D Q G E A E V K G T V S P K K 23 88 G S F L I Y P E S E A V L F S 23 99V L F S E P Q I S R G I P Q N 23 107 S R G I P Q N R P I K L L V R 23124 V V K A T N L A P A D P N G K 23 168 G E I L E L S I S L P A E T E23 9 G V N L I S M V G E I Q D Q G 22 25 A E V K G T V S P K K A V A T22 31 V S P K K A V A T L K I Y N R 22 54 F E D W L N V F P L Y R G Q G22 187 V F E H D L V G S D D L I G E 22 217 N C G L A S Q Y E V W V Q QG 22 58 L N V F P L Y R G Q G G Q D G 21 87 K G S F L I Y P E S E A V LF 21 165 P I F G E I L E L S I S L P A 20 221 A S Q Y E V W V Q Q G P QE P 20 40 L K I Y N R S L E E E F N H F 19 83 V G K F K G S F L I Y P ES E 19 121 R V Y V V K A T N L A P A D P 19 7 S D G V N L I S M V G E IQ D 18 51 F N H F E D W L N V F P L Y R 18 140 D P Y V V V S A G R E R QD T 18 155 K E R Y I P K Q L N P I F G E 18 172 E L S I S L P A E T E LT V A 18 184 T V A V F E H D L V G S D D L 18 208 N R F Y S H H R A N CG L A S 18 211 Y S H H R A N C G L A S Q Y E 18 14 S M V G E I Q D Q G EA E V K 17 36 A V A T L K I Y N R S L E E E 17 76 E E G S G H L V G K FK G S F 17 79 S G H L V G K F K G S F L I Y 17 81 H L V G K F K G S F LI Y P E 17 85 K F K G S F L I Y P E S E A V 17 122 V Y V V K A T N L A PA D P N 17 170 I L E L S I S L P A E T E L T 17 177 L P A E T E L T V AV F E H D 17 182 E L T V A V F E H D L V G S D 17 193 V G S D D L I G ET H I D L E 17 201 E T H I D L E N R F Y S H H R 17 1 M D D P G D S D GV N L I S M 16 10 V N L I S M V G E I Q D Q G E 16 13 I S M V G E I Q DQ G E A E V 16 24 E A E V K G T V S P K K A V A 16 28 K G T V S P K K AV A T L K I 16 34 K K A V A T L K I Y N R S L E 16 52 N H F E D W L N VF P L Y R G 16 57 W L N V F P L Y R G Q G G Q D 16 60 V F P L Y R G Q GG Q D G G G 16 64 Y R G Q G G Q D G G G E E E G 16 72 G G G E E E G S GH L V G K F 16 89 S F L I Y P E S E A V L F S E 16 104 P Q I S R G I P QN R P I K L 16 127 A T N L A P A D P N G K A D P 16 133 A D P N G K A DP Y V V V S A 16 163 L N P I F G E I L E L S I S L 16 171 L E L S I S LP A E T E L T V 16 174 S I S L P A E T E L T V A V F 16 186 A V F E H DL V G S D D L I G 16 192 L V G S D D L I G E T H I D L 16 195 S D D L IG E T H I D L E N R 16 6 D S D G V N L I S M V G E I Q 15 22 Q G E A E VK G T V S P K K A 15 71 D G G G E E E G S G H L V G K 15 114 R P I K L LV R V Y V V K A T 15 152 Q D T K E R Y I P K Q L N P I 15 157 R Y I P KQ L N P I F G E I L 15 166 I F G E I L E L S I S L P A E 15 181 T E L TV A V F E H D L V G S 15 HLA-DRB1*0301 (DR17) 15-mers 89 S F L I Y P E SE A V L F S E 27 199 I G E T H I D L E N R F Y S H 26 79 S G H L V G K FK G S F L I Y 25 147 A G R E R Q D T K E R Y I P K 25 156 E R Y I P K QL N P I F G E I 25 172 E L S I S L P A E T E L T V A 25 190 H D L V G SD D L I G E T H I 21 50 E F N H F E D W L N V F P L Y 20 119 L V R V Y VV K A T N L A P A 20 80 G H L V G K F K G S F L I Y P 19 107 S R G I P QN R P I K L L V R 19 113 N R P I K L L V R V Y V V K A 19 121 R V Y V VK A T N L A P A D P 19 141 P Y V V V S A G R E R Q D T K 19 160 P K Q LN P I F G E I L E L S 19 185 V A V F E H D L V G S D D L I 19 195 S D DL I G E T H I D L E N R 19 10 V N L I S M V G E I Q D Q G E 18 16 V G EI Q D Q G E A E V K G T 18 37 V A T L K I Y N R S L E E E F 18 97 E A VL F S E P Q I S R G I P 18 128 T N L A P A D P N G K A D P Y 18 217 N CG L A S Q Y E V W V Q Q G 18 12 L I S M V G E I Q D Q G E A E 17 44 N RS L E E E F N H F E D W L 17 57 W L N V F P L Y R G Q G G Q D 17 87 K GS F L I Y P E S E A V L F 17 164 N P I F G E I L E L S I S L P 17 174 SI S L P A E T E L T V A V F 17 201 E T H I D L E N R F Y S H H R 17 207E N R F Y S H H R A N C G L A 17 36 A V A T L K I Y N R S L E E E 16 51F N H F E D W L N V F P L Y R 16 142 Y V V V S A G R E R Q D T K E 16200 G E T H I D L E N R F Y S H H 16 40 L K I Y N R S L E E E F N H F 15167 F G E I L E L S I S L P A E T 15 181 T E L T V A V F E H D L V G S15 2 D D P G D S D G V N L I S M V 14 28 K G T V S P K K A V A T L K I14 47 L E E E F N H F E D W L N V F 14 96 S E A V L F S E P Q I S R G I14 209 R F Y S H H R A N C G L A S Q 14 7 S D G V N L I S M V G E I Q D13 43 Y N R S L E E E F N H F E D W 13 88 G S F L I Y P E S E A V L F S13 115 P I K L L V R V Y V V K A T N 13 116 I K L L V R V Y V V K A T NL 13 134 D P N G K A D P Y V V V S A G 13 HLA-DRB1*0401 (DR4Dw4) 15-mers113 N R P I K L L V R V Y V V K A 26 141 P Y V V V S A G R E R Q D T K26 182 E L T V A V F E H D L V G S D 26 195 S D D L I G E T H I D L E NR 26 201 E T H I D L E N R F Y S H H R 26 48 E E E F N H F E D W L N V FP 22 164 N P I F G E I L E L S I S L P 22 12 L I S M V G E I Q D Q G E AE 20 24 E A E V K G T V S P K K A V A 20 44 N R S L E E E F N H F E D WL 20 57 W L N V F P L Y R G Q G G Q D 20 80 G H L V G K F K G S F L I YP 20 88 G S F L I Y P E S E A V L F S 20 89 S F L I Y P E S E A V L F SE 20 97 E A V L F S E P Q I S R G I P 20 116 I K L L V R V Y V V K A T NL 20 119 L V R V Y V V K A T N L A P A 20 121 R V Y V V K A T N L A P AD P 20 127 A T N L A P A D P N G K A D P 20 160 P K Q L N P I F G E I LE L S 20 163 L N P I F G E I L E L S I S L 20 168 G E I L E L S I S L PA E T E 20 174 S I S L P A E T E L T V A V F 20 31 V S P K K A V A T L KI Y N R 18 36 A V A T L K I Y N R S L E E E 18 71 D G G G E E E G S G HL V G K 18 94 P E S E A V L F S E P Q I S R 18 128 T N L A P A D P N G KA D P Y 18 144 V V S A G R E R Q D T K E R Y 18 166 I F G E I L E L S IS L P A E 18 173 L S I S L P A E T E L T V A V 18 176 S L P A E T E L TV A V F E H 18 187 V F E H D L V G S D D L I G E 18 215 R A N C G L A SQ Y E V W V Q 18 222 S Q Y E V W V Q Q G P Q E P F 18 120 V R V Y V V KA T N L A P A D 17 51 F N H F E D W L N V F P L Y R 16 54 P E D W L N VF P L Y R G Q G 16 87 K G S F L I Y P E S E A V L F 16 139 A D P Y V V VS A G R E R Q D 16 185 V A V F E H D L V G S D D L I 16 207 E N R F Y SH H R A N C G L A 16 221 A S Q Y E V W V Q Q G P Q E P 16 7 S D G V N LI S M V G E I Q D 14 9 G V N L I S M V G E I Q D Q G 14 10 V N L I S M VG E I Q D Q G E 14 13 I S M V G E I Q D Q G E A E V 14 16 V G E I Q D QG E A E V K G T 14 34 K K A V A T L K I Y N R S L E 14 37 V A T L K I YN R S L E E E F 14 55 E D W L N V F P L Y R G Q G G 14 96 S E A V L F SE P Q I S R G I 14 107 S R G I P Q N R P I K L L V R 14 117 K L L V R VY V V K A T N L A 14 122 V Y V V K A T N L A P A D P N 14 156 E R Y I PK Q L N P I F G E I 14 167 F G E I L E L S I S L P A E T 14 170 I L E LS I S L P A E T E L T 14 172 E L S I S L P A E T E L T V A 14 180 E T EL T V A V F E H D L V G 14 184 T V A V F E H D L V G S D D L 14 190 H DL V G S D D L I G E T H I 14 217 N C G L A S Q Y E V W V Q Q G 14HLA-DRB1*1101 15-mers 57 W L N V F P L Y R G Q G G Q D 27 113 N R P I KL L V R V Y V V K A 22 77 E G S G H L V G K F K G S F L 21 100 L F S E PQ I S R G I P Q N R 21 201 E T H I D L E N R F Y S H H R 20 116 I K L LV R V Y V V K A T N L 19 139 A D P Y V V V S A G R E R Q D 19 167 F G EI L E L S I S L P A E T 19 221 A S Q Y E V W V Q Q G P Q E P 19 98 A V LF S E P Q I S R G I P Q 18 120 V R V Y V V K A T N L A P A D 18 207 E NR F Y S H H R A N C G L A 18 51 F N H F E D W L N V F P L Y R 16 54 F ED W L N V F P L Y R G Q G 16 58 L N V F P L Y R G Q G G Q D G 16 61 F PL Y R G Q G G Q D G G G E 16 83 V G K F K G S F L I Y P E S E 16 87 K GS F L I Y P E S E A V L F 16 118 L L V R V Y V V K A T N L A P 16 141 PY V V V S A G R E R Q D T K 16 164 N P I F G E I L E L S I S L P 16 182E L T V A V F E H D L V G S D 16 205 D L E N R F Y S H H R A N C G 16208 N R F Y S H H R A N C G L A S 16 9 G V N L I S M V G E I Q D Q G 1527 V K G T V S P K K A V A T L K 15 37 V A T L K I Y N R S L E E E F 1555 E D W L N V F P L Y R G Q G G 15 73 G G E E E G S G H L V G K F K 15149 R E R Q D T K E R Y I P K Q L 15 153 D T K E R Y I P K Q L N P I F15 79 S G H L V G K F K G S F L I Y 14 104 P Q I S R G I P Q N R P I K L14 124 V V K A T N L A P A D P N G K 14 130 L A P A D P N G K A D P Y VV 14 137 G K A D P Y V V V S A G R E R 14 163 L N P I F G E I L E L S IS L 14 195 S D D L I G E T H I D L E N R 14 6 D S D G V N L I S M V G EI Q 13 21 D Q G E A E V K G T V S P K K 13 25 A E V K G T V S P K K A VA T 13 96 S E A V L F S E P Q I S R G I 13 119 L V R V Y V V K A T N L AP A 13 160 P K Q L N P I F G E I L E L S 13 165 P I F G E I L E L S I SL P A 13 184 T V A V F E H D L V G S D D L 13 189 E H D L V G S D D L IG E T H 13 part 3: MHC Class 1115-met analysis of 158P3D2 v.3 (aa89-103-117, FRFDYLPTEREVSVRRRSGPFALEEAEFR) Pos 1 2 3 4 5 6 7 8 9 0 1 2 34 5 score HLA-DRB1*0101 15-mers 11 E V S V R R R S G P F A L E E 26 2 RF D Y L P T E R E V S V R R 19 9 E R E V S V R R R S G P F A L 18 12 V SV R R R S G P F A L E E A 17 3 F D Y L P T E R E V S V R R R 16 10 R E VS V R R R S G P F A L E 15 HLA-DRB1*0301 (DR17) 15-mers 3 F D Y L P T ER E V S V R R R 17 9 E R E V S V R R R S G P F A L 16 11 E V S V R R R SG P F A L E E 12 12 V S V R R R S G P F A L E E A 11 2 R F D Y L P T E RE V S V R R 10 10 R E V S V R R R S G P F A L E 9 8 T E R E V S V R R RS G P F A 8 HLA-DRB1*0401 (DR4Dw4) 15-mers 2 R F D Y L P T E R E V S V RR 22 3 F D Y L P T E R E V S V R R R 20 5 Y L P T E R E V S V R R R S G12 7 P T E R E V S V R R R S G P F 12 8 T E R E V S V R R R S G P F A 1215 R R R S G P F A L E E A E F R 12 HLA-DRB1*1101 15-mers 2 R F D Y L PT E R E V S V R R 25 8 T E R E V S V R R R S G P F A 21 9 E R E V S V RR R S G P F A L 21 7 P T E R E V S V R R R S G P F 20 11 E V S V R R R SG P F A L E E 12 part 4: MHC Class II 15-mer analysis of 158P3D2 v.4 (aa88-102-116, VFRFDYLPTEREVSIWRRSGPFALEEAEF) Pos 1 2 3 4 5 6 7 8 9 0 1 2 34 5 score HLA-DRB1*0101 15-mers 13 V S I W R R S G P F A L E E A 27 1 VF R F D Y L P T E R E V S I 24 12 E V S I W R R S G P F A L E E 24 10 ER E V S I W R R S G P F A L 21 3 R F D Y L P T E R E V S I W R 19 4 F DY L P T E R E V S I W R R 16 11 R E V S I W R R S G P F A L E 14HLA-DRB1*0301 (DR17) 15-mers 4 F D Y L P T E R E V S I W R R 17 10 E R EV S I W R R S G P F A L 16 12 E V S I W R R S G P F A L E E 11 13 V S IW R R S G P F A L E E A 11 3 R F D Y L P T E R E V S I W R 10 1 V F R FD Y L P T E R E V S I 9 11 R E V S I W R R S G P F A L E 9 9 T E R E V SI W R R S G P F A 8 HLA-DRB1*0401 (DR4Dw4) 15-mers 1 V F R F D Y L P T ER E V S I 22 3 R F D Y L P T E R E V S I W R 22 4 F D Y L P T E R E V SI W R R 20 13 V S I W R R S G P F A L E E A 16 10 E R E V S I W R R S GP F A L 14 6 Y L P T E R E V S I W R R S G 12 9 T E R E V S I W R R S GP F A 12 HLA-DRB1*1101 15-mers 3 R F D Y L P T E R E V S I W R 25 9 T ER E V S I W R R S G P F A 21 10 E R E V S I W R R S G P F A L 20 1 V F RF D Y L P T E R E V S I 18 12 E V S I W R R S G P P A L E E 12 part 5:MHC Class II 15-mer analysis of 158P3D2 v.5a (aa 116-178). Pos 1 2 3 4 56 7 8 9 0 1 2 3 4 5 score HLA-DRB1*0101 15-mers 30 Y Q T W C V G P G A PS S A L 26 41 S S A L C S W P A M G P G R G 26 11 V W D Y T A S L P M TS L D P 25 8 V L Q V W D Y T A S L P M T S 24 9 L Q V W D Y T A S L P MT S L 24 17 S L P M T S L D P W S C S Y Q 23 44 L C S W P A M G P G R GA I C 23 5 A V L V L Q V W D Y T A S L P 22 32 T W C V G P G A P S S A LC S 22 38 G A P S S A L C S W P A M G P 22 29 S Y Q T W C V G P G A P SS A 21 14 Y T A S L P M T S L D P W S C 20 45 C S W P A M G P G R G A IC F 20 28 C S Y Q T W C V G P G A P S S 19 31 Q T W C V G P G A P S S AL C 18 6 V L V L Q V W D Y T A S L P M 17 12 W D Y T A S L P M T S L D PW 17 48 P A M G P G R G A I C F A A A 17 3 Q P A V L V L Q V W D Y T A S16 4 P A V L V L Q V W D Y T A S L 16 35 V G P G A P S S A L C S W P A16 47 W P A M G P G R G A I C F A A 16 7 L V L Q V W D Y T A S L P M T15 24 D P W S C S Y Q T W C V G P G 14 33 W C V G P G A P S S A L C S W14 HLA-DRB1*0301 (DR17) 15-mers 3 Q P A V L V L Q V W D Y T A S 20 7 L VL Q V W D Y T A S L P M T 20 4 P A V L V L Q V W D Y T A S L 12 5 A V LV L Q V W D Y T A S L P 12 15 T A S L P M T S L D P W S C S 12 17 S L PM T S L D P W S C S Y Q 12 18 L P M T S L D P W S C S Y Q T 12 20 M T SL D P W S C S Y Q T W C 12 41 S S A L C S W P A M G P G R G 12 47 W P AM G P G R G A I C F A A 12 6 V L V L Q V W D Y T A S L P M 11 8 V L Q VW D Y T A S L P M T S 11 32 T W C V G P G A P S S A L C S 11 2 R Q P A VL V L Q V W D Y T A 10 12 W D Y T A S L P M T S L D P W 10 19 P M T S LD P W S C S Y Q T W 10 33 W C V G P G A P S S A L C S W 10 HLA-DRB1*0401(DR4Dw4) 15-mers 9 L Q V W D Y T A S L P M T S L 22 5 A V L V L Q V W DY T A S L P 20 7 L V L Q V W D Y T A S L P M T 18 33 W C V G P G A P S SA L C S W 18 38 G A P S S A L C S W P A M G P 18 11 V W D Y T A S L P MT S L D P 16 23 L D P W S C S Y Q T W C V G P 16 30 Y Q T W C V G P G AP S S A L 16 3 Q P A V L V L Q V W D Y T A S 14 4 P A V L V L Q V W D YT A S L 14 6 V L V L Q V W D Y T A S L P M 14 17 S L P M T S L D P W S CS Y Q 14 20 M T S L D P W S C S Y Q T W C 14 32 T W C V G P G A P S S AL C S 14 2 R Q P A V L V L Q V W D Y T A 12 10 Q V W D Y T A S L P M T SL D 12 12 W D Y T A S L P M T S L D P W 12 18 L P M T S L D P W S C S YQ T 12 21 T S L D P W S C S Y Q T W C V 12 24 D P W S C S Y Q T W C V GP G 12 46 S W P A M G P G R G A I C F A 12 HLA-DRB1*1101 15-mers 44 L CS W P A M G P G R G A I C 24 5 A V L V L Q V W D Y T A S L P 18 11 V W DY T A S L P M T S L D P 18 30 Y Q T W C V G P G A P S S A L 17 27 S C SY Q T W C V G P G A P S 16 17 S L P M T S L D P W S C S Y Q 14 3 Q P A VL V L Q V W D Y T A S 13 6 V L V L Q V W D Y T A S L P M 13 8 V L Q V WD Y T A S L P M T S 13 14 Y T A S L P M T S L D P W S C 12 29 S Y Q T WC V G P G A P S S A 12 32 T W C V G P G A P S S A L C S 12 38 G A P S SA L C S W P A M G P 12 41 S S A L C S W P A M G P G R G 12

[0780] TABLE XX Frequently Occurring Motifs avrg. % Name identityDescription Potential Function zf-C2H2 34% Zinc finger, C2H2 typeNucleic acid-binding protein functions as transcription factor, nuclearlocation probable cytochrome b N 68% Cytochrome b(N- membrane boundoxidase, generate terminal)/b6/petB superoxide ig 19% Immunoglobulindomain domains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored q132% NADH Membrane associated. Involved in protonUbiquinone/plastoquinone translocation across the membrane (complex I),various chains ethand 24% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha-helicaldomain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen triplehelix repeat extracellular structural proteins involved in (20 copies)formation of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeIII domain Located in the extracellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm 1 19% 7 transmembrane receptorseven hydrophobic transmembrane regions, (rhodopsin family) with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

[0781] TABLE XXI Motifs and Post-translational Modifications of 158P3D2Protein kinase C phosphorylation site 96-98 TeR 233-235 TgK Caseinkinase II phosphorylation site. 96-99 TerE 133-136 SanD 244-247 TveEAmidation site. 203-206 aGKK 255-258 kGRK Aminoacyl-transfer RNAsynthetases class-II signature .1 89-113 FRfDylpterevsvwrRsgpFaleEC2-domain. 13-142

[0782] TABLE XXII Properties of 158P3D2 Bioinformatic Variant 1 ProgramURL Outcome ORF ORF finder Protein length 328 aa Transmembrane region TMPred http://www.ch.embnet.org/ 1 TM helix 295-312 HMMTophttp://www.enzim.hu/hmmtop/ N terminus extracellular, 1TM helix aa295-314 Sosui http://www.genome.ad.jp/SOSui/ 1 TM helix 291-313 TMHMMhttp://www.cbs.dtu.dk/services/TMHMM N terminus extracellular, 1 TMhelix 292-314 Signal Peptide Signal Phttp://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW toolhttp://www.expasy.ch/tools/ 8.64 Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 38.4 kDa Localization PSORThttp://psort.nibb.ac.jp/ 85% endoplasmic reticulum, 64% peroxisome, 44%plasma membrane, 35% nucleus PSORT II http://psort.nibb.ac.jp/ 33.3%vesicles of secretory system, 22.2% cytoplasmic Motifs Pfamhttp://www.sanger.ac.uk/Pfam/ 7TM chemoreceptor Printshttp://www.biochem.ucl.ac.uk/ No significant motif Blockshttp://www.blocks.fhcrc.org/ C2 domain Bioinformatic Variant 2A ProgramURL Outcome ORF ORF finder Protein length 236 aa Transmembrane region TMPred http://www.ch.embnet.org/ no TM HMMTop http://www.enzim.hu/hmmtop/no TM, extracellular Sosui http://www.genome.ad.jp/SOSui/ no TM, solubleprotein TMHMM http://www.cbs.dtu.dk/services/TMHMM no TM Signal PeptideSignal P http://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW toolhttp://www.expasy.ch/tools/ 4.7 Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 26.1 kDa Localization PSORThttp://psort.nibb.ac.jp/ 65% cytoplasm, 10% mitochondrial matrix space,10% lysosome PSORT II http://psort.nibb.ac.jp/ 60.9% cytoplasm, 21.7%nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/ C2 domain, glutaminesynthetase Prints http://www.biochem.ucl.ac.uk/ no significant motifBlocks http://www.blocks.fhcrc.org/ C2 domain Bioinformatic Variant 2BProgram URL Outcome ORF ORF finder Protein length 181 aa Transmembraneregion TM Pred http://www.ch.embnet.org/ 1TM helix at aa 148-165 HMMTophttp://www.enzim.hu/hmmtop/ N terminus intracellular 1TM helix at aa148-167 Sosui http://www.genome.ad.jp/SOSui/ 1TM helix at aa 144-166TMHMM http://www.cbs.dtu.dk/services/TMHMM N terminus intracellular 1TMhelix at aa 148-167 Signal Peptide Signal Phttp://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW toolhttp://www.expasy.ch/tools/ 10.37 Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 21.19 kDa Localization PSORThttp://psort.nibb.ac.jp/ 85% endoplasmic reticulum, 58% peroxisome, 44%plasma membrane PSORT II http://psort.nibb.ac.jp/ 33.3% vesicles ofsecretory system, 22.2% plasma membrane Motifs Pfamhttp://www.sanger.ac.uk/Pfam/ 7TM chemoreceptor Prints http://wwwbiochem.ucl.ac.uk/ No significant motif Blockshttp://www.blocks.fhcrc.org/ no significant motif Bioinformatic Variant5A Program URL Outcome ORF ORF finder Protein length 178 aaTransmembrane region TM Pred http://www.ch.embnet.org/ N terminusextracellular, 1 TM helix 145-165 HMMTop http://www.enzim.hu/hmmtop/ Nterminus extracellular, no TM Sosui http://www.genome.ad.jp/SOSui/ noTM, soluble protein TMHMM http://www.cbs.dtu.dk/services/TMHMM Nterminus extracellular, no TM Signal Peptide Signal Phttp://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW toolhttp://www.expasy.ch/tools/ 4.49 Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 20.16 kDa Localization PSORThttp://psort.nibb.ac.jp/ 64% peroxisome, 45% cyto- plasmic, 15.3%lysosome PSORT II http://psort.nibb.ac.jp/ 52.2% cytoplasmic, 34.8%nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/ none Printshttp://www.biochem.ucl.ac.uk/ none Blocks http://www.blocks.fhcrc.org/none

[0783] Exon Number Start End TABLE XXIIIA Exon compositions of 158P3D2var1 Exon 1   1  836 Exon 2  837  922 Exon 3  923 1021 Exon 4 1022 1263Exon 5 1264 1547 Exon 6 1548 1648 Exon 7 1649 1961 TABLE XXIIIB Exoncompositions of 158P3D2 var2 Exon 1   1  95 Exon 2  96  138 Exon 3  139 239 Exon 4  240  377 Exon 5  378  494 Exon 6  495  623 Exon 7  624 1835Exon 8 1836 1921 Exon 9 1922 2020 Exon 10 2021 2222 Exon 11 2223 2506Exon 12 2507 2607 Exon 13 2608 2918

[0784] TABLE XXIIIB Exon compositions of 158P3D2 var2 Exon Number StartEnd Exon 1 1 95 Exon 2 96 138 Exon 3 139 239 Exon 4 240 377 Exon 5 378494 Exon 6 495 623 Exon 7 624 1835 Exon 8 1836 1921 Exon 9 1922 2020Exon 10 2021 2222 Exon 11 2223 2506 Exon 12 2507 2607 Exon 13 2608 2918

[0785] TABLE XXIV Nucleotide sequence of transcript variant 158P3D2atcaaggccc tgggctggag gaagacatcc cagatccaga ggagctcgac tgggggtcca 60agtactatgc gtcgctgcag gagctccagg ggcagcacaa ctttgatgaa gatgaaatgg 120atgatcctgg agattcagat ggggtcaacc tcatttctat ggttggggag atccaagacc 180agggtgaggc tgaagtcaaa ggcactgtgt ccccaaaaaa agcagttgcc accctgaaga 240tctacaacag gtccctggag gaagaattta accactttga agactggctg aatgtgtttc 300ctctgtaccg agggcaaggg ggccaggatg gaggtggaga agaggaagga tctggacacc 360ttgtgggcaa gttcaagggc tccttcctca tttaccctga atcagaggca gtgttgttct 420ctgagcccca gatctctcgg gggatcccac agaaccggcc catcaagctc ctggtcagag 480tgtatgttgt aaaggctacc aacctggctc ctgcagaccc caatggcaaa gcagaccctt 540acgtggtggt gagcgctggc cgggagcggc aggacaccaa ggaacgctac atccccaagc 600agctcaaccc catctttgga gagatcctgg agctaagcat ctctctccca gctgagacgg 660agctgacggt cgccgtattt gaacatgacc tcgtgggttc tgacgacctc atcggggaga 720cccacattga tctggaaaac cgattctata gccaccacag agcaaactgt gggctggcct 780cccagtatga agtgtgggtc cagcagggcc cacaggagcc attctgagtt tctggccaaa 840cacattcaag ctcacattcc cttttgtgtc tccagatcct atgatttcat ggaaggggac 900cctcccaccc accgccactg ccaaccaaga catagctcag tggtcaagac ttgggcttgg 960gagtcgggat cctgtaacga atgtcacttg accgctttct ttttttatga aacagtctcg 1020ctctgtctcc caggttggag tgcagtggca cgatctcggc tgactgcaac ctccacctcc 1080tgggttcaag cgattctcct gcctcagcct ccccagtagc tgggattaca ggcgtgggcc 1140cccatgtcca gctaattttt atattttcgc tctgtctccc aggttggagt gcagtggcac 1200gatctcggct gactgcaacc tccacctcct gggttcaagc gattctcctg cctcagcctc 1260cccagtagct gggattacag gcgtgggccc ccatgtccag ctaattttta tatttttagt 1320agagacaggg tttcaccatg ttgtccaggc tggtcttgaa cccctgacct caagtgatcc 1380acccacctct gcctcccaaa gtgctgggat tacaggtgtg agccaccatg ccaggccctc 1440ttaacctctt caagtctgtt ttctcatctg caaaacagag gtaataagat cagtatcttc 1500ttaatggaag cacctgggct acattttttt cattcattgt tatcataaat gaggactaac 1560ctgtctcccg ttgggagttt tgaacctaga cctcatgtct tcatgacgtc atcactgccc 1620caggcccagc tgtgtcccta caccagcccc agctgacgca tcttcttttt ctgcctgtag 1680agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 1740ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 1800agtcttcctg acaccaccgg agaccctgcc cccagggatc caagcctcgg cagccaatca 1860tctttcctca agatgtgcct gctccacccc cagttgacat caagcctcgg cagccaatca 1920gctatgagct cagagttgtc atctggaaca cggaggatgt ggttctggat gacgagaatc 1980cactcaccgg agagatgtcg agtgacatct atgtgaagag ctgggtgaag gggttggagc 2040atgacaagca ggagacagac gttcacttca actccctgac tggggagggg aacttcaatt 2100ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc gtctggcgca 2160ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg ctggtcctgc 2220aggatccctg gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg 2280ctctgtgcag ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg 2340cctgaggggc tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc 2400gcaggaggct caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga 2460cccgctgaag acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct 2700gctactggtg ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat 2760cagccaggtc atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc 2820agggtgccaa cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc 2880agagtctagt gcttcctctg aataaaccta tcacagcc 2918

[0786] TABLE XXV Nucleotide sequence alignment of 158P3D2 var1 and158P3D2 var2 Score = 2348 bits (1221), Expect = 0.0Identities= 1223/1224 (99%) Strand = Plus/Plus Query: 1tttttttatgaaacagtctcgctctgtctcccaggttggagtgcagtggcacgatctcgg 60|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1000tttttttatgaaacagtctcgctctgtctcccaggttggagtgcagtggcacgatctcgg 1059 Query:61 ctgactgcaacctccacctcctgggttcaagcgattctcctgcctcagcctccccagtag 120|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1060ctgactgcaacctccacctcctgggttcaagcgattctcctgcctcagcctccccagtag 1119 Query:121 ctgggattacaggcgtgggcccccatgtccagctaatttttatattttcgctctgtctcc 180|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1120ctgggattacaggcgtgggcccccatgtccagctaatttttatattttcgctctgtctcc 1179 Query:181 caggttggagtgcagtggcacgatctcggctgactgcaacctccacctcctgggttcaag 240|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1180caggttggagtgcagtggcacgatctcggctgactgcaacctccacctcctgggttcaag 1239 Query:241 cgattctcctgcctcagcctccccagtagctgggattacaggcgtgggcccccatgtcca 300|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1240cgattctcctgcctcagcctccccagtagctgggattacaggcgtgggcccccatgtcca 1299 Query:301 gctaatttttatatttttagtagagacagggtttcaccatgttgtccaggctggtcttga 360|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1300gctaatttttatatttttagtagagacagggtttcaccatgttgtccaggctggtcttga 1359 Query:361 acccctgacctcaagtgatccacccacctctgcctcccaaagtgctgggattacaggtgt 420|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1360acccctgacctcaagtgatccacccacctctgcctcccaaagtgctgggattacaggtgt 1419 Query:421 gagccaccatgccaggccctcttaacctcttcaagtctgttttctcatctgcaaaacaga 480|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1420gagccaccatgccaggccctcttaacctcttcaagtctgttttctcatctgcaaaacaga 1479 Query:481 ggtaataagatcagtatcttcttaatggaagcacctggactacatttttttcattcattg 540|||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| Sbjct: 1480ggtaataagatcagtatcttcttaatggaagcacctgggctacatttttttcattcattg 1539 Query:541 ttatcataaatgaggactaacctgtctcccgttgggagttttgaacctagacctcatgtc 600|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1540ttatcataaatgaggactaacctgtctcccgttgggagttttgaacctagacctcatgtc 1599 Query:601 ttcatgacgtcatcactgccccaggcccagctgtgtccctacaccagccccagctgacgc 660|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1600ttcatgacgtcatcactgccccaggcccagctgtgtccctacaccagccccagctgacgc 1659 Query:661 atcttctttttctgcctgtagagatggttacaatgcctggcgtgatgcattctggccttc 720|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1660atcttctttttctgcctgtagagatggttacaatgcctggcgtgatgcattctggccttc 1719 Query:721 gcagatcctggcggggctgtgccaacgctgtggcctccctgcccctgaataccgagccgg 780|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1720gcagatcctggcggggctgtgccaacgctgtggcctccctgcccctgaataccgagccgg 1779 Query:781 tgctgtcaaggtgggcagcaaagtcttcctgacaccaccggagaccctgcccccagggat 840|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1780tgctgtcaaggtgggcagcaaagtcttcctgacaccaccggagaccctgcccccagggat 1839 Query:841 ctcttcacatgtggattgacatctttcctcaagatgtgcctgctccacccccagttgaca 900|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1840ctcttcacatgtggattgacatctttcctcaagatgtgcctgctccacccccagttgaca 1899 Query:901 tcaagcctcggcagccaatcagctatgagctcagagttgtcatctggaacacggaggatg 960|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1900tcaagcctcggcagccaatcagctatgagctcagagttgtcatctggaacacggaggatg 1959 Query:961 tggttctggatgacgagaatccactcaccggagagatgtcgagtgacatctatgtgaaga 1020|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1960tggttctggatgacgagaatccactcaccggagagatgtcgagtgacatctatgtgaaga 2019 Query:1021 gctgggtgaaggggttggagcatgacaagcaggagacagacgttcacttcaactccctga 1080|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2020gctgggtgaaggggttggagcatgacaagcaggagacagacgttcacttcaactccctga 2079 Query:1081 ctggggaggggaacttcaattggcgctttgtgttccgctttgactacctgcccacggagc 1140|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2080ctggggaggggaacttcaattggcgctttgtgttccgctttgactacctgcccacggagc 2139 Query:1141 gggaggtgagcgtctggcgcaggtctggaccctttgccctggaggaggcggagttccggc 1200|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 11111I1III1I1I11111111111111 III111111111 111111111 1111 III Sbjct: 2140gggaggtgagcgtctggcgcaggtctggaccctttgccctggaggaggcggagttccggc 2199 Query:1201 agcctgcagtgctggtcctgcagg 1224 |||||||||||||||||||||||| Sbjct: 2200agcctgcagtgctggtcctgcagg 2223 Score = 1340 bits (697), Expect= 0.0ldentities = 697/697 (100%) Strand = Plus/Plus Query: 1263ggatccctggagttgcagctaccagacatggtgcgtggggcccggggccccgagctctgc 1322|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2222ggatccctggagttgcagctaccagacatggtgcgtggggcccggggccccgagctctgc 2281 Query:1323 tctgtgcagctggcccgcaatggggccgggccgaggtgcaatctgtttcgctgccgccgc 1382|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2282tctgtgcagctggcccgcaatggggccgggccgaggtgcaatctgtttcgctgccgccgc 2341 Query:1383 ctgaggggctggtggccggtagtgaagctgaaggaggcagaggacgtggagcgggaggcg 1442|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2342ctgaggggctggtggccggtagtgaagctgaaggaggcagaggacgtggagcgggaggcg 2401 Query:1443 caggaggctcaggctggcaagaagaagcgaaagcagaggaggaggaagggccggccagaa 1502|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2402caggaggctcaggctggcaagaagaagcgaaagcagaggaggaggaagggccggccagaa 2461 Query:1503 gacctggagttcacagacatgggtggcaatgtgtacatcctcacgggcaaggtggaggca 1562|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2462gacctggagttcacagacatgggtggcaatgtgtacatcctcacgggcaaggtggaggca 2521 Query:1563 gagtttgagctgctgactgtggaggaggccgagaaacggccagtggggaaggggcggaag 1622|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2522gagtttgagctgctgactgtggaggaggccgagaaacggccagtggggaaggggcggaag 2581 Query:1623 cagccagagcctctggagaaacccagccgccccaaaacttccttcaactggtttgtgaac 1682|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2582cagccagagcctctggagaaacccagccgccccaaaacttccttcaactggtttgtgaac 2641 Query:1683 ccgctgaagacctttgtcttcttcatctggcgccggtactggcgcaccctggtgctgctg 1742|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2642ccgctgaagacctttgtcttcttcatctggcgccggtactggcgcaccctggtgctgctg 2701 Query:1743 ctactggtgctgctcaccgtcttcctcctcctggtcttctacaccatccctggccagatc 1802|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2702ctactggtgctgctcaccgtcttcctcctcctggtcttctacaccatccctggccagatc 2761 Query:1803 agccaggtcatcttccgtcccctccacaagtgactctcgctgaccttggacactcaccca 1862|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2762agccaggtcatcttccgtcccctccacaagtgactctcgctgaccttggacactcaccca 2821 Query:1863 gggtgccaacccttcaatgcctgctcctggaagtctttcttacccatgtgagctacccca 1922|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 2822gggtgccaacccttcaatgcctgctcctggaagtctttcttacccatgtgagctacccca 2881 Query:1923 gagtctagtgcttcctctgaataaacctatcacagcc 1959||||||||||||||||||||||||||||||||||||| Sbjct: 2882gagtctagtgcttcctctgaataaacctatcacagcc 2918

[0787] TABLE XXVI Peptide sequences of protein coded by 158P3D2var2 >158P3D2 var2a MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATLKIYNRSLEEE FNHFEDWLNV 60 FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVLFSEPQISRGI PQNRPIKLLV 120 RVYVVKATNL APADPNGKAD PYVVVSAGRE RQDTKERYIPKQLNPIFGEI LELSISLPAE 180 TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGLASQYEVWVQQ GPQEPF 236 >158P3D2 var2b MVRGARGPEL CSVQLARNGA GPRCNLFRCRRLRGWWPVVK LKEAEDVERE AQEAQAGKKK 60 RKQRRRKGRP EDLEFTDMGG NVYILTGKVEAEFELLTVEE AEKRPVGKGR KQPEPLEKPS 120 RPKTSFNWFV NPLKTFVFFI WRRYWRTLVLLLLVLLTVFL LLVFYTIPGQ ISQVIFRPLH 180 K 181

[0788] TABLE XXVII Amino acid sequence alignment of 158P3D2 var1 and158P3D2 var2 Score = 372 bits (956), Expect = e − 103Identities= 181/181 (100%), Positives = 181/181 (100%) Query: 148MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 207MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK Sbjct: 1MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 60 Query:208 RKQRRRKGRPEDLEFTDMGGNVUILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 267RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS Sbjct: 61RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 120 Query:268 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 327RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH Sbjct: 121RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 180 Query:328 K 328 K Sbjct: 181 K 181

[0789]

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040003418). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A composition comprising: a substance that a) modulates the status of158P3D2, or b) a molecule that is modulated by 158P3D2 whereby thestatus of a cell that expresses 158P3D2 is modulated.
 2. A compositionof claim 1, further comprising a physiologically acceptable carrier. 3.A pharmaceutical composition that comprises the composition of claim 1in a human unit dose form.
 4. A composition of claim 1 wherein thesubstance comprises an antibody or fragment thereof that specificallybinds to a 158P3D2-related protein.
 5. An antibody or fragment thereofof claim 4, which is monoclonal.
 6. An antibody of claim 4, which is ahuman antibody, a humanized antibody or a chimeric antibody.
 7. Anon-human transgenic animal that produces an antibody of claim
 4. 8. Ahybridoma that produces an antibody of claim
 5. 9. A method ofdelivering a cytotoxic agent or a diagnostic agent to a cell thatexpresses 158P3D2, said method comprising: providing the cytotoxic agentor the diagnostic agent conjugated to an antibody or fragment thereof ofclaim 4; and, exposing the cell to the antibody-agent or fragment-agentconjugate.
 10. A composition of claim 1 wherein the substance comprisesa polynucleotide that encodes an antibody or fragment thereof either ofwhich immunospecifically bind to a 158P3D2-related protein.
 11. Acomposition of claim 1 wherein the substance comprises a 158P3D2-relatedprotein.
 12. A protein of claim 11 that is at least 90% homologous to anentire amino acid sequence shown in FIG. 2 (SEQ ID NOS: ______).
 13. Acomposition of claim 1 wherein the substance comprises a peptide ofeight, nine, ten, or eleven contiguous amino acids of FIG. 2 or Tables Vto XIX, or an analog thereof (SEQ ID NOS: ______).
 14. A composition ofclaim 1 wherein the substance comprises a CTL polypeptide of the aminoacid sequence of FIG. 2 (SEQ ID NOS: ______).
 15. A composition of claim14 further limited by a proviso that the epitope is not an entire aminoacid sequence of FIG. 2 (SEQ ID NOS:).
 16. A composition of claim 14wherein the substance comprises a CTL polypeptide set forth in Tables Vto XIX (SEQ ID NOS: ______).
 17. A composition of claim 16 furtherlimited by a proviso that the polypeptide is not an entire amino acidsequence of FIG. 2 (SEQ ID NOS: ______).
 18. A composition of claim 1wherein the substance comprises an antibody polypeptide epitope of theamino acid sequence of FIG. 2 (SEQ ID NOS: ______).
 19. A composition ofclaim 18 further limited by a proviso that the epitope is not an entireamino acid sequence of FIG. 2 (SEQ ID NOS: ______).
 20. A composition ofclaim 18 wherein the antibody epitope comprises a peptide region of atleast 5 amino acids of FIG. 2 (SEQ ID NOS: ______) in any whole numberincrement up to 328 that includes an amino acid position selected from:an amino acid position having a value greater than 0.5 in theHydrophilicity profile of FIG. 5, an amino acid position having a valueless than 0.5 in the Hydropathicity profile of FIG. 6; an amino acidposition having a value greater than 0.5 in the Percent AccessibleResidues profile of FIG. 7; an amino acid position having a valuegreater than 0.5 in the Average Flexibility profile on FIG. 8; or anamino acid position having a value greater than 0.5 in the Beta-turnprofile of FIG.
 9. 21. A composition of claim 20 further limited by aproviso that the epitope is not an entire amino acid sequence of FIG. 2(SEQ ID NOS: ______, ______, ______, ______, ______, ______, ______,______, ______, and ______).
 22. A polynucleotide that encodes a proteinof claim
 11. 23. A polynucleotide of claim 22 that comprises a nucleicacid molecule set forth in FIG.
 2. 24. A polynucleotide of claim 22further limited by a proviso that the encoded protein is not an entireamino acid sequence of FIG. 2 (SEQ ID NOS: ______).
 25. A polynucleotideof claim 22 wherein T is substituted with U.
 26. A composition of claim1 wherein the substance comprises a polynucleotide comprising a codingsequence of a nucleic acid sequence of FIG. 2 (SEQ ID NOS: ______). 27.A polynucleotide of claim 24 further comprising a polynucleotide thatencodes a 158P3D2-related protein that is at least 90% homologous to anentire amino acid sequence shown in FIG. 2 (SEQ ID NOS: ______).
 28. Acomposition comprising a polynucleotide that is fully complementary to apolynucleotide of claim
 22. 29. A composition comprising apolynucleotide that is fully complementary to a polynucleotide of claim25.
 30. A composition comprising a polynucleotide that is fullycomplementary to a polynucleotide of claim
 27. 31. A composition ofclaim 1 wherein the substance comprises a) a ribozyme that cleaves apolynucleotide having 158P3D2 coding sequence, or b) a nucleic acidmolecule that encodes the ribozyme; and, a physiologically acceptablecarrier.
 32. A composition comprising the composition of claim 1 whereinthe substance comprises human T cells, wherein said T cells specificallyrecognize a 158P3D2 peptide sequence in the context of a particular HLAmolecule.
 33. A method of inhibiting growth of cancer cells thatexpresses 158P3D2, the method comprising: administering to the cells thecomposition of claim
 1. 34. A method of claim 33 of inhibiting growth ofcancer cells that express 158P3D2, the method comprising steps of:administering to said cells an antibody or fragment thereof, either ofwhich specifically bind to a 158P3D2-related protein.
 35. A method ofclaim 33 of inhibiting growth of cancer cells that express 158P3D2, themethod comprising steps of: administering to said cells a158P3D2-related protein.
 36. A method of claim 33 of inhibiting growthof cancer cells that express 158P3D2, the method comprising steps of:administering to said cells a polynucleotide comprising a158P3D2-related protein coding sequence or a polynucleotidecomplementary to a polynucleotide having a 158P3D2 coding sequence. 37.A method of claim 33 of inhibiting growth of cancer cells that express158P3D2, the method comprising steps of: administering to said cells aribozyme that cleaves a polynucleotide having 158P3D2 coding sequence.38. A method of claim 33 of inhibiting growth of cancer cells thatexpress 158P3D2 and a particular HLA molecule, the method comprisingsteps of: administering to said cells human T cells, wherein said Tcells specifically recognize a 158P3D2 peptide subsequence in thecontext of the particular HLA molecule.
 39. A method of claim 33, themethod comprising steps of: administering a vector that delivers asingle chain monoclonal antibody coding sequence, whereby the encodedsingle chain antibody is expressed intracellularly within cancer cellsthat express 158P3D2.
 40. A method of generating a mammalian immuneresponse directed to 158P3D2, the method comprising: exposing cells ofthe mammal's immune system to a portion of a) a 158P3D2-related proteinand/or b) a nucleotide sequence that encodes said protein, whereby animmune response is generated to 158P3D2.
 41. A method of generating animmune response of claim 40, said method comprising: providing a158P3D2-related protein that comprises at least one T cell or at leastone B cell epitope; and, contacting the epitope with a mammalian immunesystem T cell or B cell respectively, whereby the T cell or B cell isinduced.
 42. A method of claim 41 wherein the immune system cell is a Bcell, whereby the induced B cell generates antibodies that specificallybind to the 158P3D2-related protein.
 43. A method of claim 41 whereinthe immune system cell is a T cell that is a cytotoxic T cell (CTL),whereby the activated CTL kills an autologous cell that expresses the158P3D2-related protein.
 44. A method of claim 41 wherein the immunesystem cell is a T cell that is a helper T cell (HTL), whereby theactivated HTL secretes cytokines that facilitate the cytotoxic activityof a cytotoxic T cell (CTL) or the antibody-producing activity of a Bcell.
 45. A method for detecting the presence of a 158P3D2-relatedprotein or polynucleotide in a sample comprising steps of: contactingthe sample with a substance of claim 1 that specifically binds to the158P3D2-related protein or polynucleotide, respectively; and,determining that there is a complex of the substance and 158P3D2-relatedprotein or the substance and 158P3D2-related polynucleotide,respectively.
 46. A method of claim 45 for detecting the presence of a158P3D2-related protein in a sample comprising steps of: contacting thesample with an antibody or fragment thereof either of which specificallybind to the 158P3D2-related protein; and, determining that there is acomplex of the antibody or fragment thereof and 158P3D2-related protein.47. A method of claim 45 further comprising a step of: taking the samplefrom a patient who has or who is suspected of having cancer.
 48. Amethod of claim 45 for detecting the presence of 158P3D2 mRNA in asample comprising: producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing 158P3D2 polynucleotides as sense and antisense primers, whereinthe 158P3D2 polynucleotides used as the sense and antisense primersserve to amplify 158P3D2 cDNA; and, detecting the presence of theamplified 158P3D2 cDNA.
 49. A method of claim 45 for monitoring 158P3D2gene products in a biological sample from a patient who has or who issuspected of having cancer, the method comprising: determining thestatus of 158P3D2 gene products expressed by cells in a tissue samplefrom an individual; comparing the status so determined to the status of158P3D2 gene products in a corresponding normal sample; and, identifyingthe presence of aberrant 158P3D2 gene products in the sample relative tothe normal sample.
 50. A method of monitoring the presence of cancer inan individual comprising: performing the method of claim 49 whereby thepresence of elevated gene products 158P3D2 mRNA or 158P3D2 protein inthe test sample relative to the normal tissue sample indicates thepresence or status of a cancer.
 51. A method of claim 50 wherein thecancer occurs in a tissue set forth in Table I.